ROLE OF CALCIUM IN ACTIVATION OF ESTROGEN RECEPTOR-α

Transcription

1 ROLE OF CALCIUM IN ACTIVATION OF ESTROGEN RECEPTOR-α A Disserttion submitted to the Fculty of the Grdute School of Arts nd Sciences of Georgetown University in prtil fulfillment of the requirements for the degree of Doctorte of Philosophy in Biochemistry nd Moleculr Biology By Shilj D Divekr Wshington DC July 30, 2008

2 Copyright 2008 by Shilj D Divekr All rights reserved ii

3 Role of clcium in ctivtion of estrogen receptor-lph Shilj D Divekr Thesis Advisor: Mry Beth Mrtin, PhD. ABSTRACT Growth fctors re thought to ctivte estrogen receptor-lph ERα by phosphorylting the serines in the N-terminus of the receptor. However, this mechnism does not ccount for the conformtionl chnges tht occur in the lignd binding domin (LBD) of receptor to render the receptor ctive. It is hypothesized tht epiderml growth fctor (EGF) ctivtes ERα through the PLC-γ-clcium-clmodulin pthwy by incresing intrcellulr clcium which, upon binding to the LBD, induces conformtionl chnge tht ctivtes the receptor. In support of this hypothesis, tretment of MCF-7 cells with EGF (150 ng/ml) led to n increse in intrcellulr clcium from 150 nm to 350 nm (+/-70 nm) nd the induction of the ERα responsive genes, progesterone receptor (PgR) nd ps2. The bility of EGF to induce PgR nd ps2 mrna ws blocked by inhibitors of phospholipse C-γ, clmodulin, nd clmodulin kinse II, the intrcellulr clcium cheltor BAPTA-AM, nd by n ntiestrogen. Tretment with high concentrtions of extrcellulr clcium lso incresed intrcellulr clcium nd induced PgR nd ps2. This induction ws blocked by chelting intrcellulr clcium providing dditionl support tht intrcellulr clcium ctivtes ERα. Tretment with clcium lso resulted iii

4 in dose dependent growth of MCF-7 cells suggesting tht clcium hs estrogen-like effects on cell prolifertion. To determine whether EGF ctivtes the C-terminus of the ERα, trnsient trnsfections were done with wild type ERα, N-terminus nd C-terminus mutnts in. As expected, EGF ctivted the wt ERα nd the N-terminus mutnt of the receptor. In ddition EGF ctivted the C-terminus mutnt suggesting tht EGF could ctivte ERα through the C-terminus. To test the hypothesis tht clcium ctivtes ERα by directly binding to the lignd binding domin of the receptor, the bility of ERα to bind to clcium ws tested. Clcium bound to the receptor with n ffinity of 0.5 µm (+/- 0.6 µm) nd four moles of clcium bound per mole of the receptor. Similr results were obtined with the LBD of the receptor suggesting tht clcium bound in the LBD of the receptor. When the LBD is ctivted, the mjor movements involve helices 3, 4, 10, 11 & 12. To determine the mino cids involved in clcium ctivtion, wild type ERα, mutnts D351A, C381A, E380Q, C447A (helix 3 & 4), mutnts E419A, E423A (helix 7 & loop between helix 7 &8), mutnts H516A, N519A, E523A (helix 10 & 11) nd mutnts N532A, D538A, E542A, D545N nd H547A (loop between helix 11&12 nd helix 12) were tested in trnsient trnsfection ssys. Of ll the mutnts tested E380 (H4), H516A (H10), E523A (H11) nd D538A (H12) significntly blocked the effect of EGF nd clcium suggesting tht these mino cids re involved in clcium ctivtion. Thus, EGF ctivtes ERα by incresing intrcellulr clcium which binds to the LBD, thereby ctivting the receptor. iv

5 Acknowledgements I would like to thnk my supervisor Dr. Mry Beth Mrtin for her guidnce, encourgement nd support for my thesis project. She hs encourged me nd helped me chieve my gols nd be good scientist. She tught me how to design experiments, write grnts nd prepred me for future s scientist. I hve lernt lot from the one to one discussions with her nd lso the lb meetings nd thnk her for this system estblished in the lb. Finlly, I would like to thnk her for understnding me when I ws going through lot of personl problems. She helped through the crisis nd lso helped me get on with my project. I once gin thnk her for her understnding, ptience nd professionlism. I lso would like to thnk Drs. Amin Fkro nd Adrin Stoic for trining me t the bench when I joined the lb. I would like to thnk other lb members Dr Dvid Veselik, Geoffery Storchn nd Dniel Prodi for being gret collegues nd friends in the lb. I would like to thnk undergrd students Ktherine Sperele nd Yusi Ljiminmuhip for working with me on this project with lots of enthusism. I would like to thnk Dr. Chetn Revnkr for her help with clcium mesurement experiments. I would like to thnk my committee members Drs. Den Rosenthl, Mrk Dnielsen, Stephen Byres nd Ngrjn Pttbirmn for their vluble suggestions, help nd time. Finlly I would like to thnk my prents for their love nd unconditionl support. I would lso like to thnk my sister, brother-in-lw nd my brother for their support. I would like to dedicte my thesis work to my lte grndmother Ms. Shntbi Divekr. She ws my role model becuse with ptience nd courge she fced ll odds in her life nd ws winner. v

6 TABLE OF CONTENTS Introduction Estrogen receptor.. 1 Functionl domins of ERα Modes of ction of ERα 3-5 Structure of ERα.. 6 Agonist induced structurl chnges. 7-9 Antgonist induced structurl chnges Hormone independent ctivtion of ERα Hormone independent ctivtion of ERα by EGF EGFR signling EGFR nd Brest cncer tretment EGFR, PLC-γ nd clcium pthwy. 21 PLC-γ nd Brest cncer Clcium Clcium binding proteins Clcium nd Brest cncer Clcium nd Clmodulin Hypothesis Results vi

7 Effect of PLC-γ inhibitor EGF induction of progesterone receptor PgR mrna 41 Effect of EGF on free intrcellulr clcium 43 Effect of EGF nd clcium pthwy inhibitors on PgR mrna expression.46 Effect of EGF nd intrcellulr clcium BAPTA-AM on ps2 mrna expression. 50 Effect of clcium on the expression of progesterone receptor nd ps2 mrna 53 Effect of extrcellulr clcium on intrcellulr clcium. 56 Effect of clcium on growth of MCF-7 cells.. 59 Effect of Cffeine on ctivity ERα in MCF-7 cells. 61 Clcium binding to recombinnt ERα Effect of EGF nd clcium on wt ERα in COS-1 cells Effect of EGF nd extrcellulr clcium on intrcellulr clcium 77 Effect of EGF on ctivtion of AF-1 nd AF-2 domins of ERα. 80 Effect of EGF on N-terminus mino cids of ERα.. 82 Effect of EGF on mino in the C-terminus mino cids Effect of clcium on the AF-1 nd AF-2 domins of the ERα. 91 Effect of clcium on mino cids in the C-terminus of ERα 95 Discussion vii

8 Mterils nd Methods Tissue Culture Regents Trnsient trnsfections nd CAT Assys Rel-time Reverse Trnscriptse-Polymerse Chin Rection RNA extrction Reverse trnscriptse-polymerse Chin Rection Rel-time Polymerse Chin Rection. 117 Clcium Mesurement Binding Assy Anchorge Dependent Growth Assy Site-Directed Mutgenesis Reference List viii

9 List of Figures Figure 1. Functionl Domins of ER... 3 Figure 2. Modes of ction of ERα 5 Figure 3: The po nd holo conformtions of ERα.. 8 Figure 4: The gonist nd the ntgonist conformtions of ERα. 11 Figure 5: EGFR medited signling pthwys. 17 Figure 6: Clcium Homeostsis Figure 7: Clcium Binding EF hnd motif 32 Figure 8: An exmple of non-ef clcium binding protein 33 Figure 9: Hypothesis- Activtion of ERα by EGF 39 Figure 10: Effect of EGF nd PLC-γ inhibitor on the expression of progesterone receptor mrna Figure 11: Effect of EGF on intrcellulr clcium.. 42 Figure 12: Effect of clcium pthwy inhibitors on PgR mrna expression Figure 13: Effect of EGF nd BAPTA-AM on ps2 mrna expression.. 48 Figure 14: Effect of clcium on ERα ctivity in MCF-7 cells.. 51 Figure 15: Effect of extrcellulr clcium on intrcellulr clcium 54 Figure 16: Effect of clcium on growth of MCF-7 cells.. 57 Figure 17: Effect of cffeine on ERα ctivity ix

10 Figure 18: Clcium binding to purified recombinnt ERα.. 62 Figure 19: Clcium binding to the lignd binding domin (LBD) of ERα 64 Figure 20: Clcium binding to purified recombinnt ERα in presence of 100x cdmium chloride. 66 Figure 21: Effect of EGF on ERα ctivity in COS-1 cells.. 70 Figure 22: Effect of clcium on ERα ctivity in COS-1 cell 71 Figure 23: Effect of EGF nd extrcellulr clcium on intrcellulr clcium. 75 Figure 24: Effect of EGF on ctivtion of the AF-1 nd AF-2 domins of ERα 78 Figure 25: Effect of EGF on the N-terminus mino cids 81 Figure 26: Effect of EGF on C-terminus mino cids Figure 27: Effect of clcium on ctivtion of the AF-1 nd AF-2 domins of ERα 90 Figure 28: Effect of clcium of ER ctivity Figure 29: Proposed model for clcium induced ction of ERα x

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12 INTRODUCTION- Estrogens regulte the growth nd differentition of the mmmry glnd 1. The biologicl effects of estrogens re medited through the estrogen receptors α nd β. Estrogen receptors [ER] re trnscription fctors belonging to the fmily of nucler receptors which lso includes ndrogen receptor, glucocorticoid receptor, progesterone receptor nd minerlocorticoid receptor 1. There re two isoforms of ER; ERα nd ERβ. Both receptors bind estrogen nd ctivte the trnscription of downstrem genes but show differences in their N-terminl regions 1. FUNCTIONAL DOMAINS OF ESTROGEN RECEPTOR α Estrogen receptors re divided into regions A through F from the N-terminus to C-terminus (Fig 1). Region A/B of the receptor hrbors the trnsctivtion function-1 (AF-1) of the receptor. The AF-1 plys n importnt role in lignd independent ctivtion of ERα. 1,2 (1, 14) It is region of the receptor involved in protein protein interctions 3, 1,4 nd phosphoryltion of specific serine residues in the A/B domin of the receptor is thought to medite lignd independent ctivtion of the receptor. Tmoxifen nd rloxifene re prtil gonist of ERα nd totl ntgonists of ERβ 5. The different effects of tmoxifen nd rloxifene on the ctivity ERα nd ERβ re ttributed to the differences in the AF-1 region of the receptor 1,5, 6. The AF-1 region of ERα s 1

13 compred to AF-1 region of ERβ plys n ctive role in the gonist nd the ntgonist effect of compounds on the receptor 1,3,7. The C region is the DNA binding domin (DBD) of the receptor. This region of the receptor binds to the estrogen response elements (ERE) on the trget genes 8. It consists of two cys-cys zinc finger motifs (CI nd CII) connected by short flexible mino cid linker 8,9. There is n mphipthic α-helix ner the C-terminus of ech finger. The first helix in the N-terminl zinc finger is clled the DNA recognition helix (CI). This helix plys n importnt role in specific DNA binding. The specificity of the DNA binding helix is due to 3 mino cids clled the P-box. Once the DBD intercts with the estrogen response element, the mino cids in the DNA recognition helix mke sequence specific interctions with nucleotides exposed on the mjor groove of the DNA. The second helix (CII) which hrbors the D-box is involved in hlf site ERE recognition nd ERα dimeriztion The miniml ERE consensus sequence is 13 bp plindromic inverted repet 5 GGTCAnnnTGACC- 3 8,9. The D region is the hinge region nd is thought to ply role in dimeriztion of the receptor 1. Following the D region is the E region. The E region is the lignd binding domin (LBD) or the hormone binding domin of the receptor. It is the region of the receptor tht is responsible for the gonistic nd ntgonistic property of vrious lignds. It hrbors the AF-2 function of the receptor nd is responsible for lignd dependent ctivtion of the receptor. It lso plys n importnt role in dimeriztion of the receptor nd the binding of coctivtors nd corepressors In the bsence of 2

14 hormones, the het shock proteins Hsp 70 nd Hsp 90 re bound in this domin of the receptor. The het shock complex renders the receptor inctive. For ERα to be ctive, the het shock proteins hve to dissocite from the receptor The role of F-region is still uncler. Brest Cncer Reserch Ruff, M et l 2 (5): FIGURE 1: Functionl Domins of ER-α. MODE OF ACTION OF ERs: There re two modes of ction of ERα. The first is the clssicl or the genomic mode of ction nd the second is the non-genomic mode of ction (Fig 2). Clssicl mode of ction: In the clssicl mode of hormone ction, upon the binding of the lignd (estrogen), the het shock complex dissocites from the estrogen receptor. The receptor then homodimerizes nd binds to estrogen response elements on the trget gene. Binding of estrdiol to the estrogen receptor lso induces conformtionl chnge in the LBD, resulting in the formtion of site for the coregultors to bind. The best 3

15 chrcterized estrogen responsive genes re progesterone receptor (PR), ps2 lso known s trefoil fctor (TIF) 19, cthepsin D, nd complement C3 8. Most estrogen response elements re orgnized s plindromic inverted repets but recently mny vrint or hlf response elements hve been identified tht re lso recognized by ERs. Exmples of vrint EREs re found in the TGFα, hsp27, nd vitllogenin A1 genes 8. Non-clssicl mode of ction: The non-clssicl mode of ction of ERα cn be divided into ERE independent genomic ctions nd non-genomic ctions. In the ERE independent genomic ctivtion, ERα ctivtes genes tht do not hve n EREs by intercting with other trnscription fctors such s AP-1 20 nd SP1 21. Estrogen receptor α binds to these trnscription fctors through protein-protein interctions nd thereby ffects their ctivity. The nongenomic ctions of ER re due to the membrne ERα which ctivtes signling pthwys such s MAPK & PI3K which in turn ctivtes downstrem genes 20,

16 Clinicl Cncer Reserch Schiff et l 9 (1): FIGURE 2: Modes of ction of ERα On binding of its estrogen lignd (E), nucler ER ctivtes trnscription (genomic ction) by directly binding to the ERE response elements on the DNA on the trget gene promoters (clssicl mode) or by binding to other trnscription fctors, such s the Fos/Jun ctivting protein-1 (AP-1) complex (nonclssicl mode). Membrne ER ctivity (nongenomic ction), re brought bout by direct interction with different signling intermedites, thereby bringing bout the rpid induction signling pthwys such s p42/44 MAPK nd phosphoinositide 3-kinse. 5

17 STRUCTURE OF ERα: The structure of full length ERα is not cler. The unstructured nture of the N- terminl A/B domin nd the inter-domin flexibility hs mde crystlliztion of the full length receptor extremely difficult 11. However the three dimensionl structure of the DBDs with n ERE nd the LBD hve been determined by crystllogrphy. The crystl structure of the LBD of the receptor hs provided useful insight into the ction of mny gonists nd ntgonists 11. The lignd binding domin of the ERα functions s moleculr switch tht upon lignd binding llows the receptor to ctivte trnscription. It consists of 11 α-helices nd short β-turn rrnged in three lyers to form n ntiprllel α-helicl sndwich (Fig 3). Helices H1-H4, H7, nd H8 form one fce of the LBD; H5, H6, H9 nd H10 nd the β-turn form the centrl lyer of the domin; nd helix H11 flnked by H12 form the second fce 11,12,26,27. The lignd binding pocket is in the lower prt of the lignd binding domin nd is closed on one side by the ntiprllel β sheet nd by helix 12 on the other side. Helix 12 is known to be directly involved in the trnsctivtion function AF-2. However, helices H3, H4, H5 re lso thought to be involved in the AF-2 function of the receptor. The position of the helix 12 is ltered by the binding of lignds. The position of helix 12 determines whether prticulr lignd is n gonist or n ntgonist 11,12,26,27. 6

18 AGONIST INDUCED STRUCTURAL CHANGES: When n gonist such s estrdiol or diethylstilbestrol (DES) is bound, the LBD of ERα undergoes conformtionl chnge from n open (po) form to closed (holo) form (Fig 3). The simplest model of conformtionl chnges tht tke plce upon lignd binding is tht helix H12 flips over the lignd binding pocket nd closes over the pocket, with the lignd trpped inside the pocket. This flipping over of helix H12 is referred s the mouse trp model. 13 However, bsed on crystl structure of po nd holo RXR-, severl dditionl structurl chnges re thought to occur in the LBD upon hormone binding; the N-terminus of helix H3 rottes pproximtely 90 0 round its xis nd bends towrds helix H4 nd the core of the hydrophobic pocket; the djcent omeg loop reloctes under helix 6; the bet turns, locted between helices H4/H5 nd H6, moves wy from the core of the pocket; helix H11 rottes pproximtely round its helicl xis nd tilts wy from the pocket thus repositioning itself long helix 10; nd helix 12 is repositioned over the LBD 12,13, It is thought tht for these structurl chnges to occur the retinoid is initilly ttrcted into the LBD by electrosttic interctions. Once inside the pocket, retinoid induces rottion of helix H3, which in turn cuses helix H11 to rotte nd form bent helix with helix H10. The repositioning of helix H11 pulls helix H12 under helix H4 freeing the omeg loop from its interction with helix H12. The stbiliztion of H12 in the gonist orienttion does not rely on direct interction between the lignd nd H12 but is dependent on the interctions mde by the lignd within the cvity 12,13,

19 TiPS Bourguet et l October2000 (Vol.21) 12 FIGURE 3: The po nd holo conformtions of ERα. In the po form helix 11 is t right ngle with helix 10 nd helix 12 is wy from the lignd binding pocket. When n gonist like estrdiol or DES is bound helix 11 rottes nd forms stright helix with helix 10 nd helix 12 flips nd closes over the pocket. This conformtionl chnge then mkes it possible for the coctivtors to bind. 8

20 Binding of coctivtors: The second essentil step fter the binding of the gonist is the binding of coctivtors. The gonist induced structurl chnges genertes the surfce to which the nucler receptor intercting domin (NID) of the coctivtors bind 12. The nucler receptor intercting domin of the coctivtors hve the co-ctivtor nucler box LxxLL motif (L: leucine, X: ny mino cid). This LxxLL motif dopts helicl conformtion nd binds to the hydrophobic groove generted by the crboxy terminl prt of the helix H3, the loop between helices H3, nd H4 nd helix H4 11,12. The helicl nture of the LxxLL motif of the coctivtor presents the first nd the third leucine residues on the sme side so tht the LxxLL motif binds in shllow depression in the hydrophobic core of the receptor. The LxxLL peptide then intercts vi its leucine residues with the hydrophobic groove nd lso by hydrogen bonds with conserved lysine (K363) t the C- terminus of the helix H3 nd glutmte (E542) in helix H12. Together these interctions form chrge clmp which stbilizes the peptide-receptor complex 11, In ddition to the stbiliztion, these interctions lso determine the length of the coctivtor motif tht cn bind to the receptor. The most chrcterized coctivtor fmily of ERα consists of SRC-1 (p160-1, N-CoA1), SRC-2 (TIF-2, GRIP1, N-CoA2), nd SRC-3 (P/CIP, ACTR, AIB1, RAC3, TRAM1). These coctivtors hve three eqully spced conserved LxxLL motifs. The other coctivtor fmily known to bind ERα is the CBP/300. They re ubiquitously expressed nd hve cetyltrnsferse ctivity 1. 9

21 ANTAGONISTS INDUCED STRUCTURAL CHANGES: When n ntgonist such s ICI 182,780 or rloxifene re bound to ERα, the LBD hs structure similr to the holo nucler receptor conformtion with one difference- helix 12 does not dopt the holo conformtion. In the presence of ntgonist, helix 12 rottes 120 o nd shifts towrds the mino terminus of the LBD. As result, helix 12 moves into the hydrophobic groove formed by helix 3, the loop L3-4 nd helix 4. This leds to helix H12 occupying the sme site where the LxxLL motifs of coctivtors bind. Thus, the ntgonist repositioning of helix 12 (AF-2 helix) prevents the formtion of the interction surfce to which coctivtors bind. The other structurl chnge tht occurs when n ntgonist is bound is with respect to helix 11. The C- terminl of the helix 11 unwinds, lengthening the loop between helices which enbles helix 12 to bind to the coctivtor LxxLL recognition cleft. In cse of ICI 182,780, the lignd side chin prevents helix 12 from dopting both the gonist nd the ntgonist position. In this cse, helix 12 does not ssocite with the LBD nd moves freely. This disordered conformtion leds to full ntgonism 12,27,28,34,35. This is one of the resons for ICI, for being complete ntgonist. 10

22 PDB Code: 3ERD 3ERR FIGURE 4: The gonist nd the ntgonist conformtions of ERα. In the DES bound conformtion the helix 12 flips over the lignd binding pocket nd cretes n interfce for coctivtors to bind. When n ntgonist like rloxifene is bound helix 12 is not ble to flip over the lignd binding pocket nd therefore coctivtors cnnot bind

23 Prtil Agonism of complete ntgonist: In ddition to full ntgonists, there re some lignds of ERα tht ct s n gonist in some tissues nd n ntgonist in others. This is often described s prtil gonism of ntiestrogens or selective estrogen receptor modultors (SERM) 12. One exmple of such lignd is tmoxifen. It cts s n ntiestrogen in the brest while cting s n gonist in bone nd other tissues. SERMS such s tmoxifen ply n importnt role in tretment of brest cncer. The mechnism of prtil gonism of n ntgonist is very complex nd not much is known. It is thought tht prtil gonism is due to the coexistence of trnscriptionlly ctive nd inctive forms of ERα when bound to lignd. A prtil gonist does not sufficiently stbilize the trnscriptionlly ctive conformtion nd permits the formtion of trnscriptionlly inctive conformtions. The shift from trnscriptionlly inctive to ctive depends on the concentrtion of trnscriptionl mchinery fctors nd the coctivtors 12,27,34, Binding of Corepressors: Corepressors by definition serve to negtively regulte the trnscriptionl ctivtion of the receptor. In the cse of the estrogen receptor, corepressors re thought to bind in the presence of n ntgonist. The best chrcterized corepressors for ERα re NCoR nd SMRT. These corepressors were initilly thought to interct with the thyroid receptor nd RXR but lter they were found to bind to most nucler receptors. Corepressors interct with ERα in presence of ntiestrogens. When helix 12 overlps 12

24 with the coctivtor interction site, it prevents coctivtor binding nd fcilittes corepressor binding. Corepressors ct by number of mechnisms to repress trnscription. Although they pper to ct predominntly through chromtin remodeling e.g. NCoR nd SMRT, corepressors compete with coctivtors for binding to the receptor 1,41. HORMONE INDEPENDENT ACTIVATION OF ERα In ddition to estrdiol, ERα is ctivted by growth fctors, such s epiderml growth fctor (EGF) 42, insulin-like growth fctor-1 (IGF-1) 42,43, nd by neurotrnsmitters such s dopmine 44. Growth fctors re thought to ctivte ERα through phosphoryltion of serines in the N-terminus (A/B domin) of the receptor. Activtion of the Rs-Rf-MEK-MAPK pthwy leds to phosphoryltion of ERα t serine S while ctivtion of the PI3K-AKT pthwy results in phosphorylting the serine ,46. However, phosphoryltion of the N-terminus does not lwys led to ctivtion of the receptor. Hormone independent ctivtion by metls- Besides growth fctors, our lb nd others hve shown tht ERα is ctivted in hormone independent mnner by bivlent ctions nd nions. These groups of estrogen receptor ctivtors re termed metlloestrogens. We hve shown tht bivlent ctions such s cdmium, coblt, nickel, nd copper nd nions such s selenite nd rsenite 13

25 ctivte ERα in the bsence of lignd. This ctivtion by bivlent ctions involves the lignd binding domin of the ERα. Bivlent ctions re thought to form high ffinity complex in the LBD of the ERα nd ctivte it. The potentil mino cids involved in metl binding hve lso been identified. For bivlent ctions such s cdmium, copper, coblt, the mino cids identified re locted on helix 4 (C381), helix 11 (E523) nd helix 12 (D538). All these helices re involved in mjor movements to induce the gonist bound conformtion 47,48. This ctivtion of ERα by metls suggests tht environmentlly relevnt doses of metls cn be potentil risk for brest cncer 49. HORMONE INDEPENDENT ACTIVATION OF ERα BY GROWTH FACTOR EGF Epiderml Growth fctor- Epiderml growth fctor is growth fctor tht plys n importnt role in the regultion of cell growth, differentition nd prolifertion. Humn EGF hs 53 mino cid residues nd three intrmoleculr disulfide binds. Epiderml growth fctor cts by binding to epidernml growth fctor receptor (EGFR) nd stimultes the intrinsic protein-tyrosine kinse ctivity of the receptor. This, in turn, ctivtes signing cscde tht results in vriety of biochemicl chnges within the cell. Epiderml growth fctor is the founding member of the EGF fmily of proteins which lso includes heprin binding EGF like growth fctor (HB-EGF), trnsforming growth fctor 14

26 (TGF-α), mphiregulin (AR), epiregulin (EPR), nd neuregulin-1 (NRG1). All the members of this fmily re very similr in structure 50,51. Epiderml growth fctor receptor (EGFR)- Epiderml growth fctor receptor (EGFR)/HER-1 or c-erbb-1 belongs to the c- erb2 fmily of receptor tyrosine kinse fmily. This fmily lso includes HER2/erbB2, HER3/erbB3 nd HER4/erbB4. Epiderml growth fctor receptor is 170-kd glycoprotein which consists of n extrcellulr domin, single trnsmembrne region nd n intrcellulr cytoplsmic region. Upon ctivtion by its lignds, EGFR dimerizes, nd undergoes utophosphoryltion of the cytoplsmic domin t tyrosine residues. The phosphoryltion of tyrosine residues results in recruitment of mny signling molecules nd downstrem signling cscdes. Epiderml growth fctor receptor cn be ctivted by EGF, TGF-α, heregulin, bet cellulin, heprin binding EGF like growth fctor, nd epiregulin 46,50,52,53. The ctivtion of erbb fmily members is tightly regulted so tht the cell cn efficiently integrte its externl stimuli with the pproprite internl function of the stimuli. This is chieved through the ctivtion of vrious signl trnsduction pthwys depending on the externl stimuli (lignd), the kind of receptor tht is expressed, nd the number of receptors tht re expressed. In cse of the erbb fmily of the receptors, the signling pthwys re not only dependent on the lignd but lso on the numerous erbb homodimer nd heterodimer combintions possible. Thus ErbB fmily of 15

27 receptors exhibits greter complexity in signling cscdes. Epiderml growth fctor receptor cn form homodimers or heterodimers with erbb2 nd erbb4 receptors. Epiderml growth fctor receptor ctivtes number of signl trnsduction pthwys such s the MAPK, STAT-3 nd PI3K/Akt pthwy. In ddition to MAPK nd PI3K pthwy EGFR cn lso ctivte the PLCγ pthwy. These signling pthwys re importnt in norml cell growth nd prolifertion 46,50,52,53. 16

28 Drug Resistnce Updtes Schmidt, M et l 2002 (5) FIGURE 5: EGFR medited signling pthwys Lignd binding induces receptor dimeriztion. Subsequent tyrosine kinse ctivity induces vrious signling pthwys including the Rs-MAPK, STAT, PLC PKC, nd PI3K-Akt pthwys which results in cellulr prolifertion, migrtion nd survivl. 17

29 EGFR SIGNALING IN BREAST CANCER EGF nd ER phosphoryltion- Epiderml growth fctor (EGF) nd EGFR signling is involved in phosphoryltion of ERα nd ctivtion of the receptor. Epiderml growth fctor nd its receptor ctivte the MAPK pthwy nd phosphorylte serine 118 in the A/B domin (N-terminus) of the ERα. Phosphoryltion of the serine 118 is thought to be involved in ctivtion of estrogen receptor nd ctivtion of ERα through EGFR signling pthwys is thought to require the A/B domin of the receptor 45. There re other serine residues in the A/B domin of the ERα tht re phosphorylted by other pthwys nmely S104/106, S154 nd S167 42,45,46, Besides the MAPK pthwy, EGF-EGFR ctivtes ERα through other signling pthwys by phosphoryltion of different serine residues in A/B domin of the receptor. The ctivtion of ERα by prticulr pthwy depends on the cell type, the second messengers vilble, nd other signling molecules. Besides EGF, IGF-1 lso ctivtes the ERα through the PI3K/AKT pthwy. This ctivtion is thought to involve the phosphoryltion of serine Phosphoryltion of coctivtors is nother mechnism by which EGFR is thought to ctivte the ERα. Growth fctor receptors increse cyclin D1 production which in turn ctivtes ERα trnscription nd intercts with coctivtor proteins such s SRC-1 nd camp response element-binding protein/p300 thereby further ctivting the ERα It hs lso been shown tht EGF signling through ERK phosphoryltes nd ctivtes 18

30 p160 GRIP1 coctivtor ctivity suggesting tht growth fctor signling to ERα cn occur in prt through coctivtor phosphoryltion 61. It is lso possible tht EGFR signling lso inhibits the ctivity of corepressors of ERα. All the dt discussed bove suggest tht EGF ctivtes ERα through the phosphoryltion of the serine residues in the N-terminus of receptor. But the ctivtion of ERα by phosphoryltion does not ccount for the conformtionl chnges tht hve to tke plce in the LBD for the receptor to be ctive. There is lso cross tlk between ERα nd EGFR. It hs been shown tht estrdiol induces EGFR nd stimultes growth of the uterus. Estrdiol induces prolifertion of uterine epithelium nd this is due to its bility to up regulte EGF. This prolifertive effect of estrdiol is blocked by EGF ntibody 62,63. Also it hs been shown in the estrogen receptor-lph knock-out mouse (ERKO), EGF induced DNA synthesis nd trnscription were bsent in uterus 64,65. Thus, it hs been shown tht there is crosstlk between ERα nd EGFR. Estrogen receptor tht is locted in the clveoli 66 of the plsm membrne induces rpid non-genomic signling nd ctivtes vriety of signling pthwy. Membrne ER signls rpidly to stimulte the trnsctivtion of EGFR leding to n increse of camp 67 nd ctivtion of ERK. Estrdiol is lso known to ctivte the mtrix metlloproteinses. Incresed mtrix metlloproteinse leds to the libertion of HB-EGF which then binds nd ctivtes EGFR 68. The membrne initited estrodiol/er signling nd its dependence on EGFR remins to be defined. Thus EGFR-ER crosstlk 19

31 is complicted nd connected t multiple levels nd plys n importnt role in progression nd tretment of brest cncer. EGFR nd BREAST CANCER TREATMENT Due to its role in the mitogenesis nd EGFR-ERα cross tlk, EGFR plys n importnt role in growth nd tretment of brest cncer. Overexpression of EGFR nd HER2 receptor hs been reported in brest cncer. There is n inverse reltionship between EGFR expression nd ER sttus in brest cncer 69. Epiderml growth fctor receptor positive nd ER negtive tumors hve the worst prognosis in tretment. Estrogen receptor positive tumors tht develop resistnce to tmoxifen re often ssocited with n increse in EGFR-HER2 signling 70. In tmoxifen resistnt ER positive brest cncer cells MCF-7, there is incresed expression of EGFR nd HER2 nd lso incresed MAPK signling 70. HER2 often hetrodimerizes with EGFR nd plys n importnt role in EGFR signling. Therefore molecules trgeting HER2 receptor in the tretment of brest cncer hve proved beneficil in tretment of the disese. Molecules, such s gefitinib tht trget the ATP binding site of the tyrosine kinse domin of EGFR, hve been shown to inhibit tumor growth of vriety of humn tumor-derived cell lines 71, lone or in combintion with other drugs or irrdition. On tretment with gefitinib, tmoxifen resistnt ER positive brest cncer cells show prolonged growth inhibition due to decrese in EGFR ctivtion nd MAPK kinse phosphoryltion 72. However these cells still develop resistnce due to 20

32 signling from IGF-1R nd PI3K 73. So combintion therpy of ntiestrogen fulvestrnt in combintion with gefinitib induces n dditive inhibition of brest cncer cell growth 50. The clinicl trget with EGFR receptors in brest cncer re still in initil stges s compred to HER2 trgeted therpy. EGFR however remins potentil therpeutic trget due to its cross-tlk with ERα nd s coreceptor for HER-2. EGFR, PLC-γ nd Clcium signling pthwy- EGFR cn lso ctivte phsopholipse C (PLC-γ) through the binding of SH2 domin of PLC-γ to phosphorylted-tyrosine sites on the EGFR receptor. Upon ctivtion, PLC-γ cleves phosphoinositol 4, 5 bisphosphte [PtdIns (4, 5) P2] to form inositol 1, 4, 5 triphosphte (IP3) nd diclyglycerol (DAG). Inositol 1, 4, 5 triphosphte nd DAG function s second messengers in the cell. Inositol 1, 4, 5 triphosphte binds to its receptor (IP3R) on the endoplsmic reticulum nd releses clcium from the endoplsmic reticulum. Clcium then functions s second messenger nd ctivtes mny proteins including clmodulin which in turn ctivtes clmodulin dependent kinses (CMK) nd other signling cscdes. Clcium nd DAG cn lso ctivte the protein kinse C (PKC) fmily of protein kinses nd consequently ctivte downstrem signling pthwys. Thus PLC-γ nd clcium signling stimulte vriety of signling pthwys nd plys n importnt role in vriety of intrcellulr functions such s mobility nd growth 52,74. 21

33 EGF, PLC-γ nd BREAST CANCER- Phospholipse C-γ belongs to fmily of phosphoinositide-specific phospholipse C enzymes nd plys n importnt role in metbolism of inositol lipids. It plys mjor role in signl trnsduction pthwys tht regulte mny cellulr processes such s cell migrtion nd motility. Upon ctivtion by growth fctors, PLC-γ is phosphorylted which then cleves its substrte phosphoinositol (4, 5) bisphosphte to IP3 nd DAG. IP3 nd DAG then ct s second messengers nd ctivte different signling cscdes PLC-γ is overexpressed in brest cncer cells nd plys n importnt role in EGFR induced tumor progression nd cell migrtion nd metstsis 74, It hs been shown tht EGF induced PLC-γ ctivtes ctin modifying enzymes which then results in rerrngement of ctin skeleton nd promotion of cell migrtion 78. Inhibition of PLC-γ by its phrmcologicl inhibitor U73122 reduces tumor cell invsion of brest cncer cells but does not ffect the prolifertion of these cells 77. It hs been proposed tht growth fctor induced ctivtion of PLC-γ nd the subsequent increse in cell invsion my be common fctor in most cncers 77. It hs been suggested tht the role of PLC-γ in cell motility nd dhesion of brest cncer MDA-231 cells fter EGF tretment my be due to its interction with PI3K 76. The role of EGFR induced ctivtion of PLC-γ is bsed on the mount of EGFR nd it heterodimer prtner c-erbb- 2 expression in cells 79. Cells over expressing these two receptor show sustined ctivtion of PLC-γ while cells expressing norml levels show short term ctivtion of 22

34 PLC-γ nd short term IP3 nd DAG turnover. This EGFR ctivtion of PLC-γ is thought to be dependent on c-erbb-2 nd c-erbb-2 up regultion, nd is responsible for switching on cell migrtion by modulting the time of PLC-γ ctivtion 79. Most of the published dt discussed bove hs been done in the more ggressive nd metsttic brest cncer cells such s MDA-231 nd MDA-HER2 nd lso in niml models with tumors. The role EGFR- PLC-γ pthwy in the neoplstic brest cncer cell line such s MCF-7 cells nd prticulrly its effects on estrogen receptor re still not very cler. It hd been shown in MCF-7 cells tht estrdiol induces phosphoryltion of PLC-γ within few seconds of tretment in MCF-7 cells nd this ctivtion is diminished fter few minutes. This ction of estrdiol is due to the nongenomic signling of estrdiol 80. It hs lso been shown in drug resistnt MCF-7 cells tht EGF ctivtes PLC-γ which then phosphoryltes p-glycoprotein which leds to decresed drug ccumultion nd incresed drug resistnce 81. In presence of EGF, physiologicl doses of linoleic cid lso ctivte PLC-γ nd PKC pthwy 82. CALCIUM Clcium (C) belongs to the tomic erth metls in the periodic tble. Its tomic weight is 20 nd tomic mss is 40. It is the fifth most bundnt element found in the erth s crust nd is found in minerls such s gypsum, clcite, nd dolomite nd is lso found in shells nd corls s clcium crbonte (CCO3)

35 The clcium ion is the most bundnt metl ion in the body. Clcium in lmost 20 times greter in terms of tom percentge in living orgnisms thn in the erths crust. Over 90% of body clcium resides in bones nd tooth enmel in the form of hydroxyptite (C 5 (PO 4 ) 3 (OH). The remining clcium is found in body fluids, blood serum, nd extrcellulr mtrix of the cell 83. The concentrtion of clcium in the extrcellulr mtrix is pproximtely 2 mm. However the intrcellulr clcium concentrtion is very low (0.1 µm) s compred to the extrcellulr clcium concentrtion. Therefore the concentrtion grdient of clcium between extrcellulr nd intrcellulr fluids is greter thn tht of N +, K + nd Mg Together the extrcellulr nd intrcellulr clcium function in mny cellulr processes, including muscle contrction, neuron communiction, neurotrnsmitter relese, hormonl relese, blood clotting, exocytosis, fertiliztion, cell dhesion, nd growth. These functions re crried out by vrious clcium chnnels, clcium pumps, nd through signling pthwys where clcium cts s second messenger to integrte the extrcellulr stimuli to the intrcellulr function. One of the most importnt functions of clcium is to function s second messenger in signling pthwys. Clcium cts s second messenger through interctions with mny proteins resulting in modultion, stbiliztion, or ctivtion of these proteins. The co-ordintion, signling, nd control of extrcellulr nd intrcellulr clcium is brought bout by vrious chnnels, receptors, clcium ATPses, nd clcium exchngers locted on the plsm membrne, the endoplsmic/srcoplsmic reticulum 24

36 nd mitochondri. The mplitude nd the frequency of clcium signls re very importnt for clcium signling. This is controlled by vriety of proteins. There re three mjor clsses of membrne-ssocited proteins involved in clcium homeostsis nmely chnnels, pumps (ATPses), nd exchngers (sodium/clcium exchngers). Clcium chnnels include voltge operted clcium chnnels (P/Q type, L-type), low voltge operted clcium chnnels (t-type), receptor operted (glutmte receptor), second messenger operted (IP3 receptors), nd store operted clcium chnnels Extrcellulr Clcium- The extrcellulr clcium is mintined between 1 to 2 mm depending upon the cell type. The mechnisms for controlling the extrcellulr clcium concentrtions re primrily dependent on extrcellulr clcium sensing receptor (CR) 86. This receptor responds to slight chnges in extrcellulr clcium. When serum clcium flls, the extrcellulr clcium sensing receptor on the prthyroid cell senses the drop in serum clcium nd enbles the cells to secrete prthyroid hormone (PTH). Prthyroid hormone relese leds to conservtion of clcium by the kidney nd incresed uptke of clcium by the intestine. These events restore serum clcium levels to norml 75,86,86. There re different mechnisms through which clcium is tken up by the cell. Voltge gted clcium chnnels trnslte the membrne depolriztion (ction potentil) into n increse in intrcellulr clcium. The lignd operted ction chnnels open on binding to n extrcellulr gonist nd increse intrcellulr clcium. Then 25

37 there re receptor-stimulted ction chnnels which re ctivted when n gonist is bound to membrne receptor distinct from the receptor-stimulted receptor. Some clcium chnnels open when the intrcellulr clcium stores (endoplsmic nd srcoplsmic reticulum) hve depleted clcium levels. These stores re clled storeoperted clcium chnnels (clcium induced clcium relese). The exct mechnism of these chnnels is not known. Clcium cn lso enter the cell through purinergic receptor nd non-selective ction chnnel 87,88. Intrcellulr Clcium- The intrcellulr clcium concentrtion is mintined t low concentrtion (0.1 µm). Most of the intrcellulr clcium is stored in the endoplsmic reticulum nd the srcoplsmic reticulum. Clcium is lso stored in the mitochondri but the min function of the mitochondri is to sequester ny hrmful increse of clcium in the cytoplsm. However, mitochondri locted in the vicinity of clcium chnnels become exposed to high clcium concentrtions nd hence temporrily store some clcium nd then relese it. The Golgi pprtus lso ccumultes clcium nd releses it when cells re exposed to IP3 87,88. 26

38 Clcium Homeostsis- The concentrtion of intrcellulr clcium in the cytoplsm is regulted by the on nd off rections. During the on rections the increse in intrcellulr clcium is due to vrious externl stimuli including growth fctors or nucleotides. This increse cn be either due to relese of clcium from intrcellulr stores such s the endoplsmic reticulum/srcoplsmic reticulum or vrious clcium chnnels, including the voltgeoperted (VOCs), receptor operted (ROCs), second messenger operted (SMOCs), or the store operted chnnels (SOCs) During the off rection, clcium is removed from the cytoplsm. This is done by the clcium ATPses locted in the endoplsmic/srcoplsmic reticulum which sequesters clcium bck into these orgnelles. Any extr intrcellulr clcium is tken up by the mitochondri vi the clcium uniporter or the sodium-clcium exchnger (NCXs) or extruded out from the cell through the NCXs nd plsm membrne clcium ATPses Increse in intrcellulr clcium is lso sequestered by proteins such s clcinurin nd clbindin which ct s clcium buffers in the cells. These proteins help to modulte clcium signling both sptilly nd temporlly s they bind free clcium to trnsmit the signl through out the cell or to remove clcium from the cytoplsm. There re other types of proteins which trnsmit the increse in intrcellulr clcium to vrious cellulr responses. These proteins function s clcium sensors (e.g. clmodulin 27

39 nd myosin) in the cell. When clcium binds it induces conformtionl chnge in the sensor. These proteins then bind to their trgets thereby ctivting them

40 Nture Reviews Moleculr Cell Biology Michel J. Berridge et l 4, (July 2003) 88 FIGURE 6: Clcium Homeostsis The on nd off rections of clcium tht tke plce in cell tht regulte clcium homeostsis. The vrious functions of clcium re lso mentioned in the figure. 29

41 CALCIUM BINDING PROTEINS- Clcium prefers to bind to oxygen thn to nitrogen nd sulfur donors. Clcium likes to form bonds dominted by ionic forces nd with low covlency. The preference for oxygen is supported by the fct tht clcium binds lignds with nitrogen donors very wekly. Most clcium binding proteins re rich in the negtively chrged mino cids sprtic cid nd glutmic cids Of these two mino cids, clcium prefers sprtic cid becuse of its less bulky side chin. The most common co-ordintion geometry of the clcium ion is seven lignds rrnged in pentgonl bipyrmidl fshion. Clcium hs the sme geometry in wter nd in most protein binding sites. In protein binding sites, this geometry is most preferred with six or seven of the chelting groups provided by the binding lignd. The co-ordintion numbers formed by clcium with decresing frequency re 8>7>6>9 92. Another unique feture of clcium bonds is tht clcium forms looser complexes of higher nd vrible coordintion number, nd with vrible bond length. Clcium coordintion is essentilly ionic nd without strong directionlity. In ddition to sprtic nd glutmic cid, clcium lso binds to crbonyl groups of sprgines nd glutmine. Clcium lso binds to phosphtes, sulftes, nd hydroxyl groups (serine) ll of which interct with clcium ions through oxygen toms 92. Most clcium binding proteins hve wht is termed s n EF hnd motif. The EF hnd is composed of helix-loop-helix structurl unit. The two helices re bridged by the clcium chelting loop. In the EF hnd, clcium hs the pentgonl bipyrmidl geometry. Clcium generlly prefers co-ordintion number of 7 in these protein motifs 30

42 91,92. In the cnonicl EF hnd, five of the seven mino cids re provided by the loop between the helices. Of the five residues, 3 to 4 bonds come from the side chin crboxy groups of the negtively chrged mino cids nd one from the crbonyl bckbone. Two of the remining bonds come from bidentte (glutmic cid) lignd which is locted in the C-terminus helix. There re non-cnonicl EF hnds where clcium binds in different wys to chieve pentgonl bipyrmidl symmetry. In some proteins such s poptosis-linked protein ALG-2, clcium binds through the octgonl symmetry rther thn the pentgonl bipyrmidl symmetry 92. There re lso clcium binding proteins tht do not hve the EF hnd symmetry. Humn mtrix metlloproteinse-26 hs two clcium binding sites. The first site (high ffinity) is composed of 3 side chin crboxy groups contributed by 2 sprttes nd one glutmte nd 3 from bckbone the crbonyl groups of glycine nd isoleucine. The second site (low ffinity) is composed of one crboxy group from sprtte nd 2 crbonyl groups from lysine nd glutmine 94. The cytosolic clcium binding domin of inositol 1,4,5-triphosphte receptor is lso not similr to EF hnd motif 94,95. Some other exmples include the bone cidic glycoprotein-75 94,96 nd epiderml growth fctor-like domin 30 of humn fibrillin-1 94,96,97 nd humn PAD4 protein (Peptidylrginine deminse 4) 98. Enzymes such s bovine pncretic deoxyribonuclese 99 nd mmmlin serum prxonses 99,100 require clcium for their ctivity. The clcium binding site in these proteins is not n EF hnd motif. 31

43 Biochem. J. (2007) 405, FIGURE 7: Clcium Binding EF hnd motif A. the occurrence of different mino cids in EF hnd clcium binding motif. B. A digrm of the C 2+ co-ordintion sphere. C. C 2+ co-ordintion by the cnonicl EFhnd (EF1 of CM) illustrting both the pentgonl bipyrmidl co-ordintion of the C 2+ ion (continuous lines) nd the extensive hydrogen bonding pttern found in the loop (broken lines)

44 NATURE STRUCTURAL & MOLECULAR BIOLOGY Kyouhei Arit1 et l VOLUME 11 NUMBER 8 AUGUST FIGURE 8: An exmple of non-ef clcium binding protein This figure shows clcium binding to the third, fourth nd fifth site on the humn PAD4 protein (Peptidylrginine deiminse 4). The left side figure shows the structure of clcium binding t the third, fourth nd fifth site. The right side shows schemtic digrm with the bond lengths. All the sites show non-ef like binding nd lso ll the sites hve co-ordintion number of 6. 33

45 CALCIUM nd BREAST CANCER - Clcium homeostsis is deregulted in brest cncer cells. In drug sensitive nd drug resistnt MCF-7 cells, clcium dynmics is different 101. The mgnitude of clcium increse ws similr upon phosphoinositol-coupled receptor gonists. However the decy kinetics of intrcellulr clcium ws much slower in drug resistnt MCF-7 cells suggesting tht intrcellulr clcium my ply role in drug resistnce 101. Polycyclic romtic hydrocrbons such s dimethylbenz[]nthrcene (DMBA) nd benzo[]pyrene (BP) re hrmful crcinogens nd re cpble of tumor initition nd progression. These compounds cuse sustined increse in intrcellulr clcium nd prolifertion of humn mmmry brest epithelil cells 102. The crcinogenic properties of these compounds my be due in prt to incresed clcium signling 102. Extrcellulr ATP nd UTP lso increse intrcellulr clcium through P2Y 2 receptor nd stimulte prolifertion of MCF-7 cells 103. Extrcellulr ATP ctivtes multiple signling pthwys including ERK/MEK kinse nd CREB resulting in ctivtion of the c-fos proto oncogene 104. Tretment with ionomycin (clcium ionophore) nd ATP hd n dditive effect on c-fos gene induction suggesting different ctivtion pthwys for extrcellulr nd intrcellulr clcium in MCF-7 cells. Extrcellulr ATP lso cts synergisticlly with EGF to ctivte c-fos suggesting cross tlk between the pthwys ctivted by ATP i.e. increse in intrcellulr clcium nd EGF 104. EGF lso enhnces clcium mobiliztion nd cpcittive clcium entry in mouse mmmry epithelil cells 103. In humn brest cncer cells, heregulin β ctivtes 34

46 store operted clcium chnnels through c-erb2 receptor 105. The ctivtion of store operted clcium chnnels is thought to be dependent on c-erb2 receptor level 105. Thus, increse in intrcellulr clcium is thought to ply n importnt role in prolifertion of brest cncer cell lines due to its bility to ctivte number of signling pthwys. Clcium chnnels, pumps in brest cncer cells Plsm membrne clcium-atpse is found in the brest cncer cell lines, MCF-7 cells nd ZR-75-1 cells 106,107. Compred to norml brest epithelil cells, these pumps re overexpressed in brest cncer cell lines 108. Inhibition of these pumps inhibits the growth of the cells suggesting role in the prolifertion of brest cncer cells 107. Not much is known bout the presence nd role of other clcium chnnels in brest cncer. However, clcium sensing receptor hs recently been found in mny brest cncer cell lines 75,109. Together with prthyroid hormone this receptor is thought to ply n importnt role in brest cncer metstses to bone. CALCIUM AND CALMODULIN- The other importnt role of clcium in ERα ctivity is through ctivtion of clmodulin. Clmodulin is thought to prticipte in ERα induced trnscriptionl ctivtion. It hs been shown tht clmodulin is required for the formtion of the ER- ERE complex nd for ctivtion of the receptor 110. Recently, it hs been shown tht clcium-clmodulin stimultes estrdiol binding to ERα 111. Clmodulin lso binds to 35

47 ERα in clcium dependent mnner nd this is thought to increse the Kd of estrdiol for receptor 112. Clmodulin is lso thought to modulte degrdtion of ERα thereby stbilizing the receptor 113,114. Thus clcium signling nd clcium binding proteins ply role in brest cncer nd in regulting ERα. 36

48 HYPOTHESIS- Estrogen receptor (ERα) belongs to the fmily of lignd inducible trnscription fctors. Upon lignd binding, ERα binds to estrogen response elements (ERE) on trget genes nd induces trnscription of trget genes. ERα plys n importnt role in the norml growth nd development of mmmry glnd. However, in brest cncer ERα, promotes the growth nd progression of the disese. The ctivity of ERα is trgeted in tretment of brest cncer. Drugs such s tmoxifen nd fulversnt re used in tretment of the disese. These compounds bind to ERα nd inhibit its ctivity. Thus, understnding the mechnism of ctivtion of ERα is of importnce in tretment of brest cncer. Upon lignd binding to ERα, het shock proteins dissocite from the receptor nd the receptor undergoes structurl chnges to form the ctive conformtion. Mjor structurl chnges include rottion of helix 3 pproximtely 90 0 round its xis, rottion of helix 11 pproximtely round its helicl xis to form continuous helix with helix 10, nd the movement of helix 12 over the lignd binding pocket. Once helix 12 is positioned over the lignd binding pocket, the coctivtors cn bind to the receptor nd bring bout the ctivtion of the receptor 27,39. Growth fctors such s epiderml growth fctor (EGF) nd insulin like growth fctor (IGF-1) re known to ctivte ERα. It is believed tht these growth fctors ctivte the receptor by phosphorylting the serine residues in the A/B domin to the receptor; i.e. S104/106, S118, nd S167 42,45,54. However, phosphoryltion of the N- 37

49 terminus does not ccount for ll the conformtionl chnges tht re required for the het shock proteins to dissocite nd for ERα to be ctive. Previously our lb hs shown tht bivlent ctions such s cdmium, copper, coblt, nd nickel ctivte ERα by forming high ffinity complex with the lignd binding domin of the receptor. It is known tht EGF increses intrcellulr clcium (bivlent ction) which in turn cts s second messenger in signling pthwys. My thesis will ddress the mechnism by which growth fctors such s EGF ctivte the ERα. My thesis will test the hypothesis tht EGF increses intrcellulr clcium, which in ddition to phosphorylting the N-terminus through signling pthwys will bind to the LBD of the receptor nd bring bout the necessry conformtionl chnges to ctivte ERα (Figure 4). To test the hypothesis, ERα positive brest cncer cell line MCF-7 will be used. The bility of BAPTA-AM, intrcellulr clcium, cheltor effect of EGF on ERα will be tested. The clcium signling pthwy involved in ctivtion of ERα by EGF will be further determined by using inhibitors of phospholipse C (U73122), clmodulin (TFP), nd PKC(H7 nd G06183). Once the pthwy is determined the bility of clcium to bind to ERα will be determined by in vitro receptor binding ssy. Muttionl nlysis will be used to determine the specific mino cids in ERα tht interct with clcium. For this, trnsient trnsfections will be done to mesure the bility of clcium to ctivte wild type ERα nd mutnt ERα. 38

50 Hypothesis EGF PIP2 PLC-γ ER Endoplmic Reticulum Phosphoryltion of N-Terminus CMK DAG IP3 CM C 2+ Direct PKC Interction??? N P P P A/B C D E F C ERα FIGURE 9- Hypothesis- Activtion of ERα by EGF. EGF ctivtes ERα by incresing intrcellulr clcium. Clcium ctivtes signling pthwys tht result in the phosphoryltion of the N-terminus nd lso binds to the lignd binding domin of the receptor resulting in conformtionl chnge tht ctivtes the receptor. Clcium my phosphorylte the receptor through the clciumclmodulin pthwy or the PKC pthwy. 39

51 RESULTS- We tested the hypothesis tht EGF ctivtes ERα by incresing intrcellulr clcium which then ctivtes signling pthwys tht result in the phosphoryltion of the N-terminus nd clcium lso binds to the lignd binding domin of the receptor resulting in conformtionl chnge tht ctivtes the receptor. To test this hypothesis the first prt of results section will focus on the increse in intrcellulr clcium by EGF tretment nd on the PLC-γ/clcium/clmodulin/clmodulin kinse pthwy. The second prt of the results will focus on the interction of clcium with ERα nd the potentil mino cids involved in the clcium interction. 40

52 Effect of PLC-γ inhibitor EGF induction of progesterone receptor mrna To determine whether epiderml growth fctor (EGF) ctivted the estrogen receptor α (ER α) through the phospholipse C-gmm pthwy (PLC-γ), the bility of EGF to induce the estrogen responsive gene progesterone receptor (PgR) ws mesured. MCF-7 cells were treted for 24 hrs with 1 nm estrdiol or 150 ng/ml of EGF in the presence or bsence of 2 µm of the PLC-γ inhibitor U73122 nd 500 nm of the ntiestrogen ICI 182,780. After 24 hrs, totl mrna ws extrcted nd the mount of PgR mrna ws determined by rel time PCR nd normlized to the mount of 18S ribosoml RNA nd expressed s percent control (Fig 10). Tretment with estrdiol resulted in n pproximtely 4-fold increse in PgR mrna tht ws blocked by the ntiestrogen but not by the PLC-γ inhibitor. Similr to estrdiol, tretment with EGF resulted in n pproximtely 3-fold induction of PgR mrna. The effect of EGF ws blocked by ntiestrogen nd by the PLC-γ inhibitor suggesting tht EGF ctivted the estrogen receptor-α through the PLC-γ pthwy. Next, we determined if there ws increse in intrcellulr clcium s result of PLC-γ ctivtion. 41

53 600 PgR mrna, %Control b 0 Control E2 E2+ICI ICI EGF EGF+ICI EGF+U73122 U73122 E2+U73122 FIGURE 10: Effect of EGF nd PLC-γ inhibitor on the expression of progesterone receptor mrna. MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency, the medi ws chnged to phenol red free IMEM supplemented with 5% CCS. After 48 hrs, the cells were treted for 24 hrs with estrdiol (1 nm) or EGF (150 ng/ml) in the presence nd bsence of the ntiestrogen ICI-182,780 (500 nm) or the PLC inhibitor U73122 (2 µm). The induction of PgR mrna ws mesured by rel time RT PCR ssy nd normlized to 18S ribosoml RNA. Dt re expressed s percent control. (men ± SD; n=3) *, p<0.05, **; p<0.005; ***, p< , compred to Control; b, compred to EGF 42

54 Effect of EGF on free intrcellulr clcium To determine whether EGF increses intrcellulr clcium downstrem of PLCγ ctivtion, MCF-7 cells were trypsinized nd incubted with the clcium dye FLUO- 3-AM for 30 min, wshed, nd resuspended in IMEM medi. MCF-7 cells were either treted with 150 ng/ml of EGF or 10 µm of ATP (Fig 11). The increse in intrcellulr clcium ws mesured in PTI fluorimeter. As expected tretment with ATP incresed intrcellulr clcium from 150 nm to 364 nm (+/- 50 nm). Similr to ATP, EGF incresed intrcellulr clcium to 350 nm (+/- 70 nm) demonstrting tht tretment of MCF-7 cells with EGF increses intrcellulr clcium. After the mesuring the increse in intrcellulr clcium we focused on clmodulin nd clmodulin kinses s downstrem ctivtors of clcium. 43

55 Reltive Fluorescence (rb) Effect of EGF on intrcellulr clcium Time [sec] ATP (10 um) EGF (150 ng/ml) FIGURE 11: Effect of EGF on intrcellulr clcium. MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48 hrs, cells were trypsinized nd 100,00,00 cells were incubted with the clcium dye FLUO 4-AM for 30 min in IMEM, wshed, nd resuspended in IMEM. EGF (150 ng/ml) nd ATP (10 µm) were dded nd the increse in intrcellulr clcium levels ws mesured in PTI fluorimeter. The dt were nlyzed using the eqution: [C 2+]= kd x ([F Fmin])/ ([Fmx F]) 115 where kd is the ffinity of the dye for clcium, F is the experimentlly mesured fluorescence, F min is the fluorescence mesured in bsence of clcium, nd F mx is the fluorescence mesured when clcium is sturted with the dye. Dt re the men from three independent experiments. 44

56 Effect of EGF nd clcium pthwy inhibitors on PgR mrna expression To determine if the ctivtion of estrogen receptor-α by EGF involved the clssicl PLC-γ- clcium pthwy, pthwy specific inhibitors were used. MCF-7 cells were treted for 24 hrs with 1nM estrdiol or 150 ng/ml of EGF in the presence or bsence of 500 nm of the ntiestrogen ICI 182,780, 1µM of the intrcellulr clcium cheltor BAPTA-AM, 1 µm of BAPTA (non-permeble clcium cheltor), 10µM of clmodulin inhibitor trifluoperzine (TFP), nd 1µM of the clmodulin kinse inhibitor KN-62. Totl mrna ws isolted nd the mount of PgR mrna ws determined by rel time RT-PCR, nd normlized to the mount of 18S ribosoml RNA, nd expressed s percent control (Fig 12 A-C). Tretment with estrdiol resulted in pproximtely 3- fold increse in PgR mrna tht ws blocked by the ntiestrogen ICI 182,780 but not by BAPTA-AM, nd TFP. EGF lso induced n pproximtely 3-fold increse in PgR mrna tht ws blocked by the ntiestrogen ICI, 182,780, BAPTA-AM, the clmodulin nd clmodulin kinse inhibitors suggesting tht EGF ctivted estrogen receptor-α by ctivting the PLC-γ-clcium pthwy. The inhibitors did not block the effect of estrdiol on ERα ctivity suggesting tht the effect on EGF ws not due to toxicity or due to direct effect on ERα. To determine whether the effect of EGF ws medited by extrcellulr clcium, BAPTA ws used s this form of BAPTA does not enter the cell. BAPTA did not block the effect of EGF suggesting tht EGF ctivtes ERα through n increse in intrcellulr clcium. 45

57 Figure 12A PgR mrna, % Control b Control E2 E2+ICI ICI EGF EGF+B-AM B-AM E2+B-AM EGF+BAPTA BAPTA Figure 12B 400 PgR mrna,%control b 0 Control E2 E2+ICI ICI 46 EGF EGF+TFP TFP E2+TFP

58 47 Figure 12 C: Control E2 E2+ICI ICI EGF EGF+KN-62(1uM) KN-62(1uM) PgR mrna,%control b Control E2 E2+ICI ICI EGF EGF+KN-62(1uM) KN-62(1uM) PgR mrna,%control Control E2 E2+ICI ICI EGF EGF+KN-62(1uM) KN-62(1uM) PgR mrna,%control b

59 FIGURE 12: Effect of clcium pthwy inhibitors on PgR mrna expression. MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48 hrs, the cells were treted for 24 hrs with estrdiol (1 nm) or EGF (150 ng/ml) in the presence nd bsence of the ntiestrogen ICI-182,780 (500 nm), the intrcellulr clcium cheltor BAPTA-AM (1 µm), the clcium cheltor BAPTA (1 µm), clmodulin inhibitor, trifluoperzine TFP (10µM) nd clmodulin kinse inhibitor KN-62 (1 µm). The induction of PgR mrna ws mesured by rel time RT PCR ssy nd normlized to 18S ribosoml RNA. Dt re expressed s percent control (men ± SD; n=3). *, p<0.05, **; p<0.005; ***, p<0.0005;. effect of clcium cheltors BAPTA-AM (B-AM) nd BAPTA; B. Effect of clmodulin inhibitor TFP; C. Effect of clmodulin kinse inhibitor KN-62., compred to control; b, compred to EGF. 48

60 Effect of EGF nd intrcellulr clcium BAPTA-AM on ps2 mrna expression To determine whether ctivtion of ERα by EGF through the clcium pthwy lso involved other estrogen responsive gene ps2, the effect of EGF nd the intrcellulr clcium cheltor BAPTA-AM on the induction of ps2 mrna ws determined. MCF-7 cells were treted for 24 hrs with 1nM estrdiol or 150 ng/ml of EGF in the presence or bsence of 500 nm ntiestrogen ICI 182,780 or 1µM of the intrcellulr clcium cheltor BAPTA-AM. Totl mrna ws isolted. The mount of ps2 mrna ws determined by rel time PCR, nd normlized to the mount of GAPDH mrna, nd expressed s percent control (Fig 13). Tretment with estrdiol resulted in n pproximtely 4.6-fold induction of ps2 mrna. Tretment with EGF resulted in n pproximtely 3-fold induction of ps2 mrna tht ws blocked by intrcellulr clcium cheltor BAPTA-AM suggesting tht EGF lso induces other estrogen responsive genes through the clcium pthwy. 49

61 Figure 13: 600 ps 2 m R N A,% C ontro b 0 Control E2 E2+ICI ICI EGF EGF+B- AM B-AM FIGURE 13: Effect of EGF nd intrcellulr clcium cheltor BAPTA-AM on ps2 mrna expression MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48 hrs, the cells were treted for 24 hrs with estrdiol (1 nm) or EGF (150 ng/ml) in the presence nd bsence of the ntiestrogen ICI-182,780 (500 nm) or the intrcellulr clcium cheltor BAPTA-AM (1 µm). The induction of ps2 mrna ws mesured by rel time RT PCR ssy nd normlized to 18S ribosoml RNA. Dt 50

62 re expressed s percent control (men ± SD; n=3). *, p<0.05, **; p<0.005; ***, p<0.0005;, compred to control; b, compred to EGF 51

63 Effect of clcium on the expression of progesterone receptor nd ps2 mrna Activtion of clcium chnnels by high concentrtions of extrcellulr clcium hs been shown to increse intrcellulr clcium 116. To determine whether ctivtion of clcium chnnels lso ctivtes the ERα by incresing intrcellulr clcium, MCF-7 cells were treted in clcium free DMEM with 5% CCS for 24 hrs with 1 nm estrdiol or 1 mm, 5 mm, or 7.5 mm clcium in the presence or bsence of 500 nm of the ntiestrogen ICI 182,780 nd 5 µm of the intrcellulr clcium cheltor BAPTA-AM. Totl mrna ws isolted. The mount of PgR nd ps2 mrna were determined by rel time PCR nd normlized to GAPDH (Fig 14). Tretment with estrdiol resulted in n pproximtely 9.8-fold nd 2.5-fold induction of PgR nd ps2, respectively. Tretment with 1 mm clcium did not increse the expression of PgR or ps2 mrna. However, tretment with 5 mm clcium resulted in n pproximtely 5-fold nd of PgR nd 3-fold of ps2 respectively. The effects of extrcellulr clcium were blocked by the ntiestrogen nd BAPTA-AM providing dditionl evidence tht increse in intrcellulr clcium ctivtes the estrogen receptor-α. Tretment with 7.5 mm clcium did not result in ny significnt ctivtion of PgR nd ps2. 52

64 Figure 14: c c b d FIGURE 14: Effect of clcium on ERα ctivity in MCF-7 cells. MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency, the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48, hrs the cells were treted in clcium free DMEM with 5% CCS for 24 hrs with estrdiol (1 nm) or clcium (1.0, 5.0 nd 7.5 mm) in the presence nd bsence of the ntiestrogen ICI-182,780 (500 nm) nd the intrcellulr clcium cheltor BAPTA-AM (1 µm). The induction of PgR nd ps2 mrna were mesured by rel time RT PCR ssy nd normlized to GAPDH. Dt re expressed s percent control. (men ± SD; n=3). *, p<0.05, **; p<0.005; ***, p< , compred to control 53

65 (PgR); b, Compred to EGF (PgR); c, compred to control (ps2); d, compred to EGF (ps2). 54

66 Effect of extrcellulr clcium on intrcellulr clcium To show tht high concentrtions of extrcellulr clcium incresed intrcellulr clcium, MCF-7 cells were treted with the clcium dye FLUO-3-AM for 30min, wshed, trypsinized, nd resuspended in IMEM. MCF-7 cells were either treted with 10 µm of ATP or 5 mm of clcium. The increse in intrcellulr clcium ws mesured in PTI fluorimeter (Fig 15). As expected, ATP incresed intrcellulr clcium from 150 nm to 380 nm (+/-65). Similr to ATP, 5 mm extrcellulr clcium incresed intrcellulr clcium from 165 nm to 500 nm (+/- 100 nm). 55

67 Figure 15: Effect of clcium on intrcellulr clcium Reltive Fluorescence (rb) Time (sec) C (5 mm) ATP (10 um) FIGURE 1: Effect of extrcellulr clcium on intrcellulr clcium. MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency, the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48 hrs, cells were trypsinized nd 100,00,00 cells were incubted with clcium dye FLUO 4-AM for 30 min in IMEM, wshed, nd resuspended in IMEM. The effects on intrcellulr clcium were mesured in the presence of extrcellulr clcium (5 mm) or ATP (10 µm) in PTI fluorimeter. The dt were nlyzed using the eqution: [C 2+]= kd x ([F Fmin])/ ([Fmx F]) 115 where kd is the ffinity of the dye for clcium, F is the experimentlly mesured fluorescence, F min is the fluorescence 56

68 mesured in bsence of clcium, nd F mx is the fluorescence mesured when clcium is sturted with the dye. Dt re the men from three independent experiments. 57

69 Effect of clcium on growth of MCF-7 cells To determine whether clcium hs estrogen like effects on growth, the bility of clcium to replce estrdiol in the hormone dependent prolifertion of MCF-7 cells ws tested. Cells were treted with 1 nm of estrdiol or 0.25, 0.5, 1.0 nd 2 mm of clcium on dy 0 in the presence nd bsence of 500 nm of the ntiestrogen ICI 182,780 (Fig 16A). On dy 4, the totl number of cells ws counted using Coulter counter. Tretment with estrdiol resulted in n pproximtely 3.8-fold induction of growth while tretment with clcium resulted in n pproximtely 2-fold induction of growth. MCF-7 cells were then treted with 0.1, 0.25, 0.5 nd 1.0 mm of clcium on dys 0, 1, 2 nd 3 nd cells were counted on dy 4 (Fig 16B). Tretment with estrdiol resulted in n pproximtely 3.1-fold increse in cell number. Tretment with clcium resulted in n pproximtely 2- to 3.5-fold increse in cell number nd showed dose dependent effect with incresing concentrtions of extrcellulr clcium. The bility of the ntiestrogen to inhibit clcium induced cell growth suggests tht the effects of clcium re medited by ERα. 58

70 Figure 16A: Cell number,% Control Control E2 E2+ICI ICI C(0.25) C(0.25)+ICI C(0.5) C(0.5)+ICI C(1) C(1)+ICI C(2) C(2)+ICI Figure 16B: Treted everydy Cell number,% Control Control E2 E2+ICI ICI C(0.1) C(0.1)+ICI C(0.25) C(0.25)+ICI C(0.5) C(0.5)+ICI C(1) C(1)+ICI 59

71 FIGURE 16: Effect of clcium on growth of MCF-7 cells. MCF-7 cells were plted in phenol red IMEM supplemented with 5% CCS. After 48 hrs, the medi ws chnged to phenol red free IMEM supplemented with 5% CCS. The cells were treted with estrdiol (1 nm) or clcium (0.1, 0.25, nd 0.5, 1.0 nd 2 mm) on dy 0 in presence nd bsence of the ntiestrogen ICI-182,780 (500 nm). The totl number of cells ws counted on dy 4 using the coulter counter (Fig 16A). MCF-7 cells were lso treted with clcium (0.1, 0.25, 0.5, 1.0 mm) on dys 0, 1, 2 nd 3 in the presence nd bsence of the ntiestrogen, ICI 183, 720 (500 nm) nd cells were counted on dy 4 (Fig 16B). (men ± SD; n=3) *, p<0.05, **; p<0.005; ***, p< , compred to control 60

72 Effect of Cffeine on ctivity ERα in MCF-7 cells Cffeine is known to increse intrcellulr clcium by blocking the reuptke of clcium by clcium-atpses in the srcoplsmic reticulum 117. To determine if increse in intrcellulr clcium by other stimulus such s cffeine lso ctivtes ERα, MCF-7 cells were treted with estrdiol (1 nm) or different concentrtions of cffeine ( , 5.0, 10.0 mm) in the presence or bsence of the ntiestrogen ICI, 182,780 (500 nm) nd intrcellulr clcium cheltor BAPTA-AM (1 µm) (Fig 17). Tretment with estrodiol resulted in n pproximtely 5.7-fold induction in PgR mrna. Tretment with 5 mm nd 10 mm cffeine resulted in n pproximtely 2- nd 2.4-fold induction of PgR mrna, respectively, tht ws blocked by both the ntiestrogen nd the intrcellulr clcium cheltor suggesting tht cffeine induced ctivtion of ERα by incresing intrcellulr clcium. 61

73 Figure 17: PgR mrna,%control b b Control E2 E2+ICI ICI Cffeine (1) Cffeine (2) Cffeine (5) Cf+B-AM Cf(5)+ICI Cffeine (10) Cf(10) +B-AM Cf(10)+ICI FIGURE 17: Effect of cffeine on ERα ctivity. MCF-7 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency, the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48 hrs, the cells were treted for 24 hrs with estrdiol (1 nm) or cffeine (1.0, 2.0, 5.0 nd 10 mm) in the presence nd bsence of the ntiestrogen ICI-182,780 (500 nm) or the intrcellulr clcium cheltor BAPTA-AM (1 µm). The induction of PgR mrna ws mesured by rel time RT PCR ssy nd normlized to GAPDH. Dt re expressed s percent control. (men ± SD; n=3) *, p<0.05, **; p<0.005; ***, p< , compred to control. b, compred to cffeine. 62

74 Clcium binding to recombinnt ERα The second prt of the hypothesis ws to determine if clcium ctivtes ERα by directly intercting with the lignd binding domin of the receptor. To determine if clcium binds to ERα, receptor binding ssys were done. Recombinnt humn ER- (375 x moles) ws incubted with vrious concentrtions of 45 C (10-8 M, 0.25 x 10-8 M, 0.5 x 10-8 M, 0.75 x 10-8 M, 10-7 M,.25 x 10-7 M, 0.5 x 10-7 M, 0.75 x 10-7 M, 10-6 M, 1.5 x 10-6 M, 2 x 10-6 M nd M) in the presence nd bsence of 100-fold molr excess of CCl 2 to determine non-specific nd totl binding respectively. The dissocition constnt for clcium binding ws determined by non-liner regression nlysis nd ws found to be pproximtely 0.5 x 10-6 M (± 0.6 x 10-6 M, n = 3) (Fig 18 A & B). Lignd binding domin (LBD) of the estrogen receptor α ws used to determine whether clcium binds to this domin of the receptor. In this ssy, purified recombinnt lignd binding domin (513 x moles) of ER- ws incubted with vrious concentrtions of 45 C (10-8 M, 0.25 x 10-8 M, 0.5 x 10-8 M, 0.75 x 10-8 M, 10-7 M,.25 x 10-7 M, 0.5 x 10-7 M, 0.75 x 10-7 M, 10-6 M, 1.5 x 10-6 M, 2 x 10-6 M nd M) in the presence nd bsence of 100-fold molr excess of CCl 2 to determine the non-specific nd totl binding respectively. The dissocition constnt for clcium binding to the LBD is pproximtely 0.5 x 10-6 M (±0.5 x 10-6 M, n = 3) (Fig 19 A & B). When the dt ws plotted s moles bound per mole of the receptor, the lignd binding domin bound 4 moles of 45 C per mole of receptor. 63

75 To determine whether other bivlent ctions cn function s cold competitors, estrogen receptor binding ssys were done in the presence of excess cdmium. Recombinnt humn ER- (375 x moles) ws incubted with vrious concentrtions of 45 C ( M, 0.25 x 10-8 M, 0.5 x 10-8 M, 0.75 x 10-8 M, 10-7 M,.25 x 10-7 M, 0.5 x 10-7 M, 0.75 x 10-7 M, 10-6 M, 1.5 x 10-6 M, 2 x 10-6 M nd M) in the presence nd bsence of 100-fold molr excess of CdCl 2. Cdmium competed with clcium for binding. The dissocition constnt for clcium, determined by nonliner regression nlysis, nd ws pproximtely 1.6 x 10-6 M (±0.5 x 10-6 M, n = 3) (Fig 20 A & B). 64

76 Figure 18A: Specific Binding (cpm) C-45 binding to recombinnt ER Dt Conc (um) Clcium (um) Specific binding (cpm) Totl binding Specific binding Non-specific binding Figure 18B: C-45 binding to recombinnt ER Specific Binding (cpm) Clcium (um) Specific binding 65

77 FIGURE 18: Clcium binding to purified recombinnt ERα. Purified recombinnt ERα ws diluted in the rection buffer to concentrtion of x10-15 moles per rection. Rdioctive 45 C ( x 10-6 M) ws dded to ech rection in the bsence (totl binding) nd the presence of 100x excess of cold clcium ( x 10-4 M, non-specific binding.) The rection ws incubted overnight t 4 0 C to rech sturtion. Dextrn coted chrcol ws dded to ech rection nd free 45 C ws removed by centrifugtion. Bound 45 C ws mesured by scintilltion counter. The dt were nlyzed using non-liner regression nlysis. The dt shown re representtive of 3 independent experiments. A. Specific binding of clcium plotted on liner scle. B. Specific binding of clcium plotted on log scle (semi-logrithmic plot) 66

78 Figure 19A: Specific binding (cpm) Clcium-45 binging to LBD of ER Clcium (um) C-45 binding C-45 binding to LBD of ER conc Specific binding totl binding non-specific binding Figure 19B: C-45 binding to LBD of ER 3000 Specific binding Specific binding Clcium (um) 67

79 FIGURE 19: Clcium binding to the lignd binding domin (LBD) of ERα. Lignd binding domin of ERα ws diluted in the rection buffer to concentrtion of 557 x10-15 moles per rection. Rdioctive 45 C ( x 10-6 M) ws dded to ech rection in the bsence (totl binding) nd the presence of 100x excess of cold clcium ( x 10-4 M, non-specific binding.) The rection ws incubted overnight t 4 0 C to rech sturtion. Dextrn coted chrcol ws dded to ech rection nd free 45 C ws removed by centrifugtion. Bound 45 C ws mesured by scintilltion counter. The dt were nlyzed using non-liner regression nlysis. The dt shown re representtive of 3 independent experiments. A. Specific binding of clcium plotted on liner scle. B. Specific binding of clcium plotted on log scle (semi-logrithmic plot) 68

80 Figure 20A: Clcium-45 binding to recombinnt ER in presence of 100x excess cdmium 4000 Specific binding Specific Binding Clcium (um) Figure 20B: Clcium-45 binding to recombinnt ER in presence of 100x excess cdmium 4000 Specific binding Specific Binding Clcium (um) 69

81 FIGURE 20: Clcium binding to purified recombinnt ERα in presence of 100x cdmium Purified recombinnt ERα ws diluted in the rection buffer to concentrtion of x10-15 moles per rection. Rdioctive 45 C ( x 10-6 M) ws dded to ech rection in the bsence (totl binding) nd the presence of 100x excess of cold cdmium ( x 10-4 M, non-specific binding.) The rection ws incubted overnight t 4 0 C to rech sturtion. Dextrn coted chrcol ws dded to ech rection nd free 45 C ws removed by centrifugtion. Bound 45 C ws mesured by scintilltion counter. The dt were nlyzed using non-liner regression nlysis. The dt shown re representtive of 3 independent experiments. A. Specific binding of clcium plotted on liner scle. B. Specific binding of clcium plotted on log scle (semi-logrithmic plot) 70

82 Effect of EGF nd clcium on wt ERα in COS-1 cells To identify the potentil mino cids involved in binding to clcium we tested wild type nd mutnts of ERα in trnsient trnsfection ssys. First we tested the bility of EGF nd clcium to ctivte wild-type ERα in COS-1 cells. To test the bility of EGF to ctivte ERα, trnsient trnsfection ssys were done. COS-1 cells were trnsiently cotrnsfected with wild type ERα, n estrogen response element-cat reporter construct nd β-glctosidse. Trnsfected cells were then treted for 24 hrs with 1nM estrdiol nd 150 ng/ml of EGF in the presence nd bsence of 500 nm of the ntiestrogen ICI 182,780 nd 2 µm of the PLC-γ inhibitor U73122 (Fig 21). CAT ctivity ws mesured nd normlized to β-glctosidse ctivity, nd expressed s percent control. Tretment with estrdiol resulted in n pproximtely 3.6-fold increse in CAT ctivity tht ws blocked by the ntiestrogen ICI 182,780. Similrly, EGF resulted in pproximtely 4-fold induction increse in CAT ctivity tht ws blocked by both ICI 182,780 nd U73122 suggesting tht EGF ctivted ERα through the PLC-γ pthwy. As negtive control, COS-1 cells were trnsiently cotrnsfected with the glucocorticoid receptor (GR) nd MMTV-CAT construct nd treted with 10 nm of dexmethsone, 1 nm of estrdiol nd 150 ng/ml of EGF. As expected, tretment with dexmethsone resulted in 7-fold induction of CAT ctivity while tretment with estrdiol nd EGF did not induce CAT ctivity. To test the bility of clcium to ctivte ERα in COS-1 cells, optimum time nd dose ws first determined. COS-1 cells were trnsiently cotrnsfected with wild type 71

83 ERα, estrogen response element-cat reporter construct, nd β-glctosidse. COS-1 cells were then treted for 6, 12 nd 24 hours with 1 nm estrdiol nd 0.5, 1 nd 2 mm of clcium. The mount of CAT ctivity ws mesured, normlized to the mount of β- glctosidse ctivity, nd expressed s percent control (Fig 22A). Tretment with estrdiol for 12 nd 24 hours gve 1.7- nd 2.8-fold induction respectively. Tretment of clcium for 12 hours gve 1.7-, 2.1- nd 1.4-fold induction t 0.5, 1 nd 2 mm, respectively. Tretment of clcium for 24 hours gve 1.7-, 2.1- nd 2-fold induction t 0.5, 1 nd 2 mm, respectively. Tretment t 6 hours did not result in ny induction with both estrdiol nd clcium. Bsed on these results, further clcium experiments were done t dose of 1 mm for 24 hours. To test the specificity of clcium to ctivte ERα, COS-1 cells were trnsiently cotrnsfected with wild type ERα, n estrogen response element-cat reporter construct nd β-glctosidse or the glucocorticoid receptor (GR) nd the glucocorticoid responsive MMTV-CAT construct. Trnsfected cells were then treted for 24 hrs with 1 nm estrdiol, 10 nm of dexmethsone nd 0.5 mm, 1mM nd 2 mm of clcium in the presence nd bsence of 500nM of the ntiestrogen ICI 182,780. The mount of CAT ctivity ws mesured nd normlized to the mount of β-glctosidse ctivity. The results re expressed s percent control (Fig 22B). Tretment with estrdiol resulted in pproximtely 3-fold induction of CAT ctivity. Tretment with 0.5 mm, 1 mm, nd 2 mm resulted in pproximtely in 1.7-fold, 3-fold nd 2-fold induction of CAT ctivity, 72

84 respectively, tht ws blocked by ICI 182,780 suggesting tht clcium ctivted ERα. In contrst to ERα, GR ws not ctivted by clcium. 73

85 Figure 21: 800 CAT Activity % Contro b c w/o wter wt ER GR 0 Control E2 E2+ICI ICI EGF EGF+ICI EGF+U73122 U73122 DEX FIGURE 21: Effect of EGF on ERα ctivity in COS-1 cells. COS-1 cells were plted in phenol red free IMEM supplemented with 5% CCS nd were trnsiently co-trnsfected with the wt ERα, GR, CAT reporter gene, nd β-gl. The cells were treted for 24 hours with estrdiol (1 nm) nd EGF (150 ng/ml), with or without ntiestrogen ICI 182,780 (500 nm). The effect of EGF on ERα ctivity ws mesured s the mount of CAT ctivity, normlized to the mount of β-glctosidse ctivity, nd expressed s the percentge control. (men ± SD, n=3). ) *, p<0.05, **; p<0.005; ***, p< , compred to control; b, compred to EGF; c, compred to EGF. 74

86 Figure 22A: 300 CAT Activity,%Control hrs 12 hrs 24 hrs Control ICI E2 E2+ICI C(0.5) C(0.5)+ICI C(1) C(1)+ICI C(2) C(2)+ICI C(5) C(5)+ICI Figure 22B: 800 CAT Activity % Control b b w/o wterα wterα GR Control E2 E2+ICI ICI C(0.5) C(0.5+ICI) C(1) C(1+ICI) C(2) C(2)+ICI DEX 75

87 FIGURE 22: Effect of clcium on ERα ctivity in COS-1 cells. COS-1 cells were plted in phenol red free IMEM supplemented with 5% CCS nd were trnsiently co-trnsfected with the wt ERα, GR, CAT reporter gene, nd β-gl. The cells were treted for 6, 12 nd 24 hours (Fig 22A) or 24 hours (Fig 22B) with estrdiol (1 nm), clcium (0.5, 1.0, 2, nd 5 mm) with or without the ntiestrogen ICI 182,780 (500 nm). The effect of clcium on ERα ctivity ws mesured s the mount of CAT ctivity, normlized to the mount of β-glctosidse ctivity, nd expressed s percent control. A. Time course of clcium tretment. B. Effect of clcium on ERα ctivity. (men ± SD, n=3). *, p<0.05, **; p<0.005; ***, p< , compred to control; b, compred to EGF 76

88 Effect of EGF nd extrcellulr clcium on intrcellulr clcium To determine whether tretment of COS-1 cells with EGF nd extrcellulr clcium increses intrcellulr clcium, the cells were loded with FLUO-3 dye for 30 minutes. The cells were wshed, trypsinized, nd resuspended in medi. Cells were then treted with EGF (150 ng/ml), clcium (1 mm), or ATP (10 µm). The increse in intrcellulr clcium ws mesured in PTI fluorimeter (Fig 23). As expected, tretment with ATP resulted in increse in intrcellulr clcium from 100 nm to 350 nm (+/- 75). Similr to ATP, tretment with EGF nd clcium resulted in n increse in intrcellulr clcium from 120 nm to 400 nm (+/- 100) nd 500 nm (+/- 75), respectively. 77

89 Figure 23: Effect of EGF nd clcium on intrcellulr clcium Reltive fluorescence (rb) Time COS-1 ATP COS-1 EGF COS-1 C(1mM) FIGURE 2: Effect of EGF nd extrcellulr clcium on intrcellulr clcium. COS-1 cells were plted in phenol red IMEM supplemented with 5% FBS. At 60% confluency, the medi ws chnged to phenol red free IMEN supplemented with 5% CCS. After 48 hrs, cells were trypsinized nd 100, 00, 00 cells were incubted with clcium dye FLUO-3-AM for 30 min in IMEM. The cells were then wshed nd resuspended in IMEM. EGF (150 ng/ml), extrcellulr clcium (1 mm), nd ATP (10 µm) were dded nd intrcellulr clcium ws mesured using PTI fluorimeter. The dt were nlyzed using the eqution: [C 2+]= kd x ([F Fmin])/ ([Fmx F]) 115 where kd is the ffinity of the dye for clcium, F is the experimentlly mesured 78

90 fluorescence, F min is the fluorescence mesured in bsence of clcium, F mx is the fluorescence mesured when clcium is sturted with the dye. The results re the men of two independent experiments. 79

91 Effect of EGF on ctivtion of AF-1 nd AF-2 domins of ERα To identify the region of ERα tht is ctivted by EGF, trnsient trnsfections were conducted using mutnts of the ERα (Fig 24A). To determine whether EGF ctivted through the N-terminus, the HE15 construct contining the A/B, C, nd prt of D domin of the receptor ws used. To determine whether EGF ctivted through the C-terminus, the HE19 construct contining the DNA binding domin nd the lignd binding domin ws used. COS-1 cells were trnsiently cotrnsfected with truncted ERα mutnts nd n estrogen responsive-cat reporter construct. The cells were treted for 24 hrs with 1 nm estrdiol nd 150 ng/ml of EGF in the presence nd bsence of 500 nm of ICI 182,780. The mount of CAT ctivity ws mesured, normlized to the mount of β-glctosidse ctivity, nd expressed s percent control. In COS-1 cells trnsfected with wt ERα, estrdiol nd EGF tretment resulted in pproximtely 2.5-fold nd 3-fold increse in CAT ctivity, respectively. When cells were trnsfected with the N-terminus of ERα nd treted with estrdiol, there ws no increse in the CAT ctivity s expected. However, tretment with EGF resulted in 2- fold increse in CAT ctivity suggesting tht EGF increses the ctivity of the N- terminus of the receptor. When cells were trnsfected with the C-terminus of ERα, tretment with estrdiol nd EGF resulted in n pproximtely 3-fold nd 2-fold induction of CAT ctivity, respectively, tht ws blocked by ntiestrogen (Fig 24B). These results suggest tht, in ddition to the N-terminus, EGF ctivtes ERα through the lignd binding domin of the receptor. 80

92 Figure 24A: wt Figure 24B: 400 CAT Activity % Control wt HEG15 HE19 0 Control E2 E2+ICI ICI EGF EGF+ICI 81