IGF-I and IGFBP-3 augment transforming growth factor-b actions in human renal carcinoma cells

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1 originl rticle & Interntionl Society of Nephrology IGF-I nd IGFBP-3 ugment trnsforming growth fctor-b ctions in humn renl crcinom cells AH Rosendhl 1, nd G Forsberg 1 Deprtment of Oncology, Institution of Clinicl Sciences, Lund University, University Hospitl, Lund, Sweden nd Active Biotech Reserch AB, Lund, Sweden Renl cell crcinom (RCC) is the most prevlent cncer of the kidney. In humn RCC cells, we recently showed tht insulin-like growth fctor I (IGF-I) hs growth-promoting effects regulted by IGF-binding protein 3 (IGFBP-3). In this study, the nlysis ws expnded to include the interction between the IGF nd trnsforming growth fctor-b (TGF-b) systems in the humn RCC cells Cki- (from primry tumor) nd SK-RC-5 (from metstsis). Functionl effects such s cell prolifertion, TGF-b receptor (TbR) signling, nd IGFBP-3 levels were monitored fter stimultion with vrious concentrtions of IGF-I, TGF-b, nd IGFBP-3. In ddition, humn RCC tissues s well s experimentl humn RCC tumors were nlyzed for cellulr expression of phosphorylted Smd by immunohistochemistry. TGF-b regulted the endogenous IGFBP-3 levels in these RCC cells s neutrlizing nti-tgf-b(1 3) ntibodies strongly reduced the bsl IGFBP-3 level. In ddition, IGF-I incresed the IGFBP-3 levels five- to eightfold with TGF-b cting in synergy to enhnce the IGFBP-3 levels 1- to 17-fold. Neutrlizing TGF-b(1 3) ctivity circumvented the growth inhibitory effects of IGFBP-3 seen in SK-RC-5, wheres it inhibited the growth-promoting effects of IGFBP-3 in Cki-. Moreover, IGF-I intercted directly with TGF-b ctivtion of the TbR complex by enhncing phosphoryltion nd nucler trnsloction of Smd. This study demonstrtes direct interction of the IGF nd TGF-b systems in humn renl crcinom cells. The observtions tht IGF-I enhnces the TGF-b signling nd tht TGF-b promotes IGFBP-3 production nd thus influence the biologicl ctivity of IGF my be of importnce for future therpeutic options. Kidney Interntionl () 7, doi:1.13/sj.ki.515; published online 13 September KEYWORDS: IGF; IGFBP-3; TGF-b; Smd phosphoryltion; neutrliztion; renl cell crcinom Correspondence: G Forsberg, Active Biotech Reserch AB, Box 7, S- 7 Lund, Sweden. E-mil: gorn.forsberg@ctivebiotech.com Received 17 October 5; revised 1 April ; ccepted My ; published online 13 September Renl cell crcinom (RCC) is highly vsculrized tumor nd the most frequent cncer of the kidney. Almost 5% of the ptients develop metstsis, nd the prognoses for these ptients re reltively poor. The mechnisms responsible for tumor growth nd progression of RCC re uncler; however, the von Hippel Lindu (VHL) tumor suppressor gene is mutted in most ptients with spordic cler-cell RCC, 1 which leds to utocrine dysregultion of severl functions including vsculr endothelil growth fctor, trnsforming growth fctor-b (TGF-b), nd insulin-like growth fctor (IGF) ctions. Like severl other forms of cncer, RCC cn exhibit insulin-like growth fctor I (IGF-I)-dependent growth through the type I IGF tyrosine kinse receptor. 5 7 Retrospective studies suggest tht type I IGF tyrosine kinse receptor expression in the primry tumor is ssocited with poor survivl of ptients with erly-stge RCC nd high pretretment IGF-I levels my limit the efficcy of interleukin- immunotherpy in dvnced cncer ptients. 9 In ddition, incresed gene expression nd plsm levels of IGF-binding protein 3 (IGFBP-3) nd TGF-b1 re commonly ssocited with RCC tumors. 1,11 IGFBP-3 is the min crrier of IGFs in the circultion, where this complex prolongs the hlf-life of the IGFs nd regultes their biologicl response. 1 Accumulting evidence suggests tht the IGFBP-3 plys n importnt role in the regultion of both cell growth nd deth, independently of its interction with the IGFs. 13,1 TGF-b is multifunctionl cytokine tht belongs to lrge fmily of structurlly homologous dimeric proteins. TGF-b exhibits brod rry of biologicl effects including immunosuppression, regultion of extrcellulr mtrix formtion, stimultion of ngiogenesis, s well s growth inhibitory or stimultory effects depending on trget cell. 15 The biologicl response to TGF-b involves cscde of interdependent events from the cell surfce serine/ threonine kinse receptors to the nucleus. TGF-b binds the constitutively ctive type II TGF-b receptor (TbRII), which then phosphorylte the type I TGF-b receptor. Downstrem signls re propgted by ctivted nd phosphorylted Smd nd Smd3, which ssocite with the common meditor Smd nd re trnslocted to the nucleus where they prticipte in the regultion of trnscription of trget genes Kidney Interntionl () 7,

2 AH Rosendhl nd G Forsberg: IGF-I nd IGFBP-3 in humn RCC originl rticle In mlignncies such s brest cncer, IGFBP-3 nd TGF-b cn interct with ech other s signling pthwys nd it hs been suggested tht they my regulte ech other s cellulr effects. TGF-b1 increses the secretion of IGFBP-3 in vriety of brest cncer cell lines. 17,1 In turn, IGFBP-3 potentites the TGF-b effects by enhncing the phosphoryltion of Smd nd Smd3. Moreover, inhibitory IGFBP-3 signling hs been suggested to require n ctive TGF-b signling pthwy nd the presence of TGF-b nd TbRII. 19, In this study, the cross-tlk between the TGF-b superfmily nd IGF system in humn RCC ws investigted. Furthermore, we exmined the functionl effects of TGF-b1 on IGFBP-3 production nd cell growth modultion, s well s the effects of IGF-I nd IGFBP-3 induction of TbR complex signling by phosphoryltion of Smd. [S] b RESULTS Phosphorylted Smd in humn RCC tissue indictes n ctive TbR signling The TGF-b system hs role in the pthogenesis of severl humn cncers, mnifested in incresed serum levels of TGFb1 nd impired or ltered TbR signling. 1,15,1 To exmine if there re indictions of ctive TGF-b signling pthwys in humn renl cncer, tissues from four cler cell, one ppillry, s well s four experimentl xenogrft tumors of Cki- cells or one of SK-RC-5 cells were nlyzed by immunohistochemistry for the expression of TbRs nd Smd proteins. A strong immunorectivity of phosphorylted Smd ws observed in the mjority of tumor cells, wheres n intermedite stining ws observed in vsculr endothelil cells from both humn RCC tissue (Figure 1) nd xenogrft Cki- (Figure 1b) nd SK-RC-5 tumors (Figure 1c). Importntly, these cells displyed significnt nucler ccumultion of psmd, indicting n ongoing signling. In shrp contrst, neither cytoplsmic nor nucler psmd ws detected in the surrounding stroml cells in the humn RCC tissue (Figure 1). Type I TGF-b receptor/ ALK-5 ws expressed on tumor, strom, nd endothelil cell. A homogeneous immunorection of TbRII ws shown on the tumor nd endothelil cells, wheres strom nd subendothelil cells showed weker rection. A similr immunostining ws detected for Smd, Smd3, nd Smd signling proteins (dt not shown). The immunorections ppered similr mong the tumor smples nlyzed, nd the xenogrft RCC tumors showed similr immunostining of ll TbR signling members s the humn tumor tissues. These results show the presence of ll members essentil to form the TbR complex in humn RCC. Most importntly, the mjority of the tumor cells, in contrst to stroml cells, trnsduce n ctive TbR signling monitored s nucler trnsloction of psmd. Effects of TGF-b1 on growth control TGF-b1 is known to medite either inhibitory or stimultory effects on cell growth of vrious tumors. The growthmodulting effects of TGF-b1 in the presence or bsence of c Figure 1 Cellulr distribution of phosphorylted Smd in humn RCC tissue nd xenogrft RCC tumors. Cler-cell RCC cryosections ( mm) were immunostined for psmd s described in the Mterils nd Methods section. The expression is visulized in brown by diminobenzidine þ nd cells counterstined by methyl green. Tumor cells in () humn cler-cell RCC tissue, (b) xenogrft Cki-, nd (c) xenogrft SK-RC-5 tumors showed positive nucler stining of psmd s indicted by rrows. No immunorectivity ws observed in the surrounding stroml cells (S) in the () humn RCC tissue. (, c) Br ¼ 5 mm or(b) 1 mm. The illustrtions show one representtive tissue from ech tumor type from experiments repeted t lest three times. IGF-I nd IGFBP-3 were investigted in vitro. High concentrtions of exogenous TGF-b1 hd smll stimulting effect on Cki- cell growth in the bsence of other growth fctors (Figure ). Tretment with constnt concentrtions of IGF-I or combintion of IGF-I nd IGFBP-3 strongly enhnced the cell growth; however, ddition of low doses of TGF-b1 (1 ng/ml) inhibited the prolifertive response to IGF-I nd to IGF-I with IGFBP-3 by 11 nd 3%, respectively. Further, incresed TGF-b1 concentrtions (1 ng/ml) lmost restored the prolifertion rte to control levels. In contrst, recombinnt TGF-b1 inhibited the bsl cell growth of SK-RC-5 cells with decrese in prolifertion of % t 5 ng/ml s compred to control (Figure b). The suppressive effect of TGF-b1 ws further enhnced in the presence of IGF-I or the combintion of IGF-I nd IGFBP-3, where 5 ng/ml TGF-b1 decresed the prolifertive response to IGF-I by % nd to IGF-I with IGFBP-3 by 57% (Figure b). Thus, TGF-b ffects the prolifertive ctivity differently in these RCC cells. IGF-I nd IGFBP-3 seem to sensitize the cells to growth inhibitory effects of TGF-b1. TGF-b1 tretment increses IGFBP-3 levels In ddition to TGF-b1, IGFBP-3 levels re often elevted in RCC ptients nd my ply role in pthogenesis. 11 To investigte the effects of IGF-I nd TGF-b1 on the regultion of IGFBP-3 levels, serum-free conditioned medium from treted Cki- nd SK-RC-5 cells were collected nd nlyzed for IGFBP-3 content (Tble 1). Bsl levels of Kidney Interntionl () 7,

3 originl rticle AH Rosendhl nd G Forsberg: IGF-I nd IGFBP-3 in humn RCC IGFBP-3 were in the order of ng/ml. TGF-b1 tretment incresed the IGFBP-3 protein level threefold in Cki- cells nd sevenfold in SK-RC-5 cells. In ddition, the IGFBP-3 level ws enhnced fivefold for Cki- nd eightfold for SK-RC-5 fter IGF-I tretment (Pp.1). Concomitnt tretment with IGF-I nd TGF-b1 hd synergistic effect on IGFBP-3 levels, which were incresed 1-fold (Cki-) or 17-fold (SK-RC-5) (Pp.1). In contrst, neutrlizing TGF-b(1 3) strongly reduced the bsl level of IGFBP-3 by 3% in Cki- nd % in SK-RC-5 (Pp.1). These findings demonstrte the involvement of TGF-b in the regultion of IGFBP-3 protein levels, resulting in tight interction of their respective functionl cellulr responses TGF-β1 concentrtion (ng/ml) b TGF-β1 concentrtion (ng/ml) Figure Differentil effects of TGF-b on Cki- nd SK-RC-5 cell growth in vitro. Prolifertion determined by 3 H-thymidine uptke of () Cki- nd (b) SK-RC-5 cells cultured h in SFM (white brs) or in SFM contining IGF-I (3 ng/ml) (blck brs) or IGF-I (3 ng/ml) þ IGFBP-3 (5 ng/ml) (gry brs) nd incresing concentrtions of TGF-b1. Grph represents the men of three experiments ech repeted in triplicte. Effects of IGFBP-3 on cell growth fter TGF-b(1 3) neutrliztion The involvement of utocrine TGF-b on the prolifertive responses to IGF nd/or IGFBP-3 ws investigted by dding neutrlizing TGF-b(1 3) monoclonl ntibody (mab) to the cells under serum-free conditions. An excess of neutrlizing TGF-b(1 3) ntibody dose dependently decresed the IGF-I-, IGFBP-3-, or IGF-I/IGFBP-3-induced prolifertion rte of Cki-, with mximum inhibition of 3% t mg/ml with simultneous tretment with IGFBP-3 or IGF-I/IGFBP-3 (Figure 3). In contrst, neutrlizing ny endogenous TGF-b ctivity in SK-RC-5 cells using the neutrlizing TGF-b(1 3) ntibody dose dependently enhnced the bsl prolifertion, with mximum increse of % t mg/ml s compred to control (Figure 3b). However, the cell growth fter neutrlizing endogenous TGF-b(1 3) ws not further ffected by the presence of exogenous IGF-I, IGFBP-3, or the two combined. Thus, TGF-b displys disprte effects on the bsl growth of these RCC cells. These dt suggest n effect of endogenously produced TGF-b on the IGF-I- nd IGFBP-3-medited utoor prcrine cell growth of Cki- cells in vitro. IGF-I induces Smd phosphoryltion in Cki- cells As mesurement of ctive TGF-b signling, the induction nd phosphoryltion pttern of Smd in Cki- or SK-RC- 5 cells by TGF-b, nd the impct by IGF-I nd IGFBP-3, ws nlyzed in vitro using semiquntittive immunocytostining. For Cki-, very low bsl level of psmd ws detected in untreted control cells (Figure ). Tretment with exogenous TGF-b1 only resulted in wek induction of Smd phosphoryltion (Figure b). Interestingly, concomitnt tretment with IGF-I strongly enhnced this phosphoryltion nd distinct nucler locliztion ws observed, demonstrting full onset of TGF-b-regulted genes (Figure e). Notbly, the Smd phosphoryltion fter IGF- I tretment ws still strongly enhnced even in the bsence of exogenous TGF-b1 (Figure d) nd fter ntibody neutrliztion of endogenous TGF-b(1 3) (Figure f), lthough the psmd levels were reduced compred to the combintion of IGF-I nd TGF-b1 (Figure e) nd were pproximtely equl to IGF-I lone (Figure d). Furthermore, IGFBP-3 tretment enhnced the phosphoryltion of Smd. However, concomitnt incubtion with IGF-I reduced the levels of 1 1 SFM IGF-I IGFBP-3 IGF-I + IGFBP-3 b 1 SFM IGF-I IGFBP-3 IGF-I + IGFBP-3 Figure 3 TGF-b neutrliztion countercts IGFBP-3 cellulr effects. Prolifertion determined by 3 H-thymidine uptke of () Cki- nd (b) SK-RC-5 cells cultured h in SFM, SFM contining IGF-I (3 ng/ml), or IGF-I (3 ng/ml) in combintion with IGFBP-3 (5 ng/ml) in the presence of neutrlizing nti-tgf-b(1 3) mab mg/ml (white brs), 1 mg/ml (blck brs), or mg/ml (gry brs). Grph represents the men of three experiments ech repeted in triplicte. Tble 1 IGF-I nd TGF-b1 synergisticlly induce IGFBP-3 expression in RCC cells IGFBP-3 (ng/ml) Cell line Bsl expression IGF-I stimultion TGF-b1 stimultion IGF-I+TGF-b1 stimultion TGF-b(1 3) neutrliztion Cki SK-RC IGF-I, insulin-like growth fctor I; IGFBP-3, IGF-binding protein 3; RCC, renl cell crcinom; TGF-b1, trnsforming growth fctor-b. IGFBP-3 levels in 1 ml conditioned medium from RCC cells grown for 7 h in SFM in the presence or bsence of IGF-I (3 ng/ml), TGF-b1 (5 ng/ml), or neutrlizing nti-tgfb(1 3) Ab ( mg/ml) were mesured by ELISA ssy s described under Mterils nd Methods. Results re shown s the mens7s.e. of six replicte wells from one of three independent experiments. The vrious tretments were sttisticlly significnt (Pp.1) reltive to bsl IGFBP-3 expression. 15 Kidney Interntionl () 7,

4 AH Rosendhl nd G Forsberg: IGF-I nd IGFBP-3 in humn RCC originl rticle SFM TGF-β1 Anti-TGF-β(1-3) b c SFM IGF-I Figure Enhnced induction nd nucler trnsloction of phosphorylted Smd in Cki- cells by IGF-I stimultion. Cki- cells treted 15 min in ( c) SFM or (d f) SFM in the presence of IGF-I 3 ng/ml, (b, e) TGF-b1 5 ng/ml or (c, f) neutrlizing nti-tgf-b(1-3) mab mg/ml were immunostined for phosphorylted Smd (. mg/ml) nd shown t originl mgnifiction. The expression is visulized in brown by diminobenzidine nd cells counterstined by methyl green. Green rrows indicte positive nucler stining for psmd, wheres red rrows show negtive stining. One representtive experiment out of two. psmd. A similr reduction of IGFBP-3-induced psmd ws noted fter endogenous TGF-b(1 3) neutrliztion (dt not shown). As for Cki-, very wek bsl level of psmd ws detected in untreted SK-RC-5 cells. The stining ws minly cytoplsmic nd only few cells demonstrted nucler trnslocted psmd. Incubtion with IGF-I lone or in the combintion with TGF-b1 did not ffect the Smd phosphoryltion to degree significntly different from the bsl level. As the stining intensity ws very wek, no conclusive results could be obtined for the ctivtion of Smd fter TGF-b1 or IGF-I stimultion in SK-RC-5 (dt not shown). These results suggest tht differentil signling pthwys re ctivted upon TGF-b tretment in these cells. Furthermore, the results show potentil signling crosstlk between the IGF nd TGF-b systems in Cki- cells, where IGF-I is ble to enhnce downstrem TbR signling pthwys through the induction of psmd. DISCUSSION TGF-b nd IGFBP-3 levels re incresed in RCC ptients. 1,11 However, their roles in disese progression re poorly understood. We investigted the responsiveness to TGF-b nd interctions between the TbR nd IGF receptor signling d e f pthwys in one primry tumor nd one medistinl metstsis RCC cell line. The results demonstrte tht these signling pthwys indeed re coupled in these RCC cells. TGF-b influences the levels of IGFBP-3, thus regulting its intrinsic effects s well s the effects of IGFs. Moreover, IGF-I nd IGFBP-3 influence the TGF-b signling by enhncing both phosphoryltion nd nucler trnsloction of Smd. TGF-b-induced growth inhibition of the SK-RC-5 metstsis cell line ws ccompnied by concomitnt increse in endogenous IGFBP-3 levels. In these cells, IGFBP- 3 cts s negtive regultor nd suppress cellulr growth. The sensitivity to TGF-b-induced growth inhibition ws further enhnced by co-tretment with exogenous IGFBP-3 nd TGF-b. Neutrliztion of TGF-b reduced the IGFBP-3 levels, which led to enhnced bsl prolifertion nd prtilly rescued SK-RC-5 cells from TGF-b nd IGFBP-3 growthinhibitory effects. These results re consistent with previously described brest cncer systems, where TGF-b-induced growth inhibition t lest prtly occurs through n incresed production of IGFBP-3 17 nd where IGFBP-3 cn ctivte the TGF-b signling pthwy by enhncing the phosphoryltion of Smd nd Smd3, thus incresing the sensitivity to TGF-b growth-modulting effects. 19, TGF-b lso incresed IGFBP-3 levels in Cki- cells. However, in shrp contrst to SK-RC-5, IGFBP-3 enhnces prolifertion in this primry tumor cell. In line with this, TGF-b tretment slightly stimulted cell growth, wheres neutrliztion of TGF-b reduced the bsl IGFBP-3 production nd subsequently inhibited the prolifertion. Thus, our results suggest tht role of TGF-b in RCC cells is to influence the production nd modulte the effects of IGFBP-3. Wheres TGF-b1-medited growth inhibition of Cki- ws only reveled in the presence of exogenous IGF-I, TGFb1 suppressed the bsl growth of SK-RC-5, lthough the growth inhibitory properties of TGF-b1 ppered to be more pronounced in the presence of IGF-I. Thus, one lterntive wy to interpret our dt would be tht TGF-b inhibition occurs vi inhibition of IGF-medited growth nd tht the different responses to TGF-b ctions on the bsl growth in these cells my be result of differences in their utocrine IGF production. However, Cki-, in which bsl growth ws not suppressed by TGF-b1, produces low levels of IGF-II, wheres no utocrine IGFs hve been detected in the highly TGF-b-responsive SK-RC-5. Thus, the observtion tht TGF-b inhibition ws enhnced in the presence of exogenous IGF-I suggests tht IGF-I sensitizes the cells to the growth inhibitory properties of TGF-b. TGF-b comprises both tumor suppressor nd tumor promoter functions, which re likely to be medited by differences in the ctivtion of vrious downstrem TbR signling pthwys. However, it remins uncler during which stge of tumor progression nd through which moleculr mechnisms this pleiotrophic cytokine switches its role from tumor suppressor to tumor promoter. A loss of function or expression of the TbRII is observed in severl Kidney Interntionl () 7,

5 originl rticle AH Rosendhl nd G Forsberg: IGF-I nd IGFBP-3 in humn RCC tumors, including metsttic RCCs. 1 The functionl loss of TGF-b responsiveness nd thus loss of TGF-b-medited growth suppression hs been shown to be ssocited with n incresed risk of trnsformtion. 15 Primry tumors my reduce or lter their response to TGF-b in order to fcilitte invsiveness nd metsttic potentil. 15, Furthermore, when the metstsis reches its secondry site, it my gin dopt more primry tumor-like phenotype from TGF-b perspective. Exogenous TGF-b1 only modertely ffected the growth properties of Cki-, which my be owing to reduced or ltered sensitivity to TGF-b signling. This ws further supported t the receptor signling level where the bsl psmd level ws very low, nd exogenous TGF-b1 only mrginlly induced phosphoryltion nd nucler trnsloction of Smd, hllmrk of ctive TGF-b signling. Intrinsic ctivities of IGFBP-3 ws previously described to include signling through the Smd pthwy, where IGFBP-3-stimulted phosphoryltion of Smd nd Smd3 in T7D-RII nd MCF-7 brest cncer cells. We lso confirmed tht exogenous IGFBP-3 incresed the phosphoryltion s well s nucler trnsloction of Smd in Cki- cells. However, simultneous incubtion with exogenous IGF-I reduced the levels of psmd. This might be owing to complex formtion of IGF-I:IGFBP-3, limiting free IGFBP-3 to exert its biologicl ctions nd induce signling. Interestingly, IGF-I lone or in combintion with TGF-b1 enhnced both the phosphoryltion nd distinct trnsloction to the nucleus of psmd, indicting ctive receptor signling. This enhnced phosphoryltion occurs fter 1 min of IGF-I stimultion, which renders it unlikely to be owing to n incresed IGFBP-3 production. Our current findings re consistent with recent publiction where tretment of prosttic epithelil cells with TGF-b1 nd LR 3 -IGF-I (IGF-I nlog with reduced ffinity for most IGFBPs) incresed the psmd levels over the levels induced by TGF-b lone. 3 Interestingly, lthough SK-RC-5 cells re highly responsive to TGF-b1-medited growth inhibition, the bsl levels of phosphorylted Smd were low nd minly cytoplsmic. Within the limits of the ssy, further tretment with exogenous TGF-b1 or IGF-I did not significntly lter the phosphoryltion or nucler trnsloction of Smd s compred with untreted cells. This indictes tht other signling pthwys my be mediting the growth inhibitory effects of TGF-b1onin vitro cultured SK-RC-5 cells. Thus, the different responses to TGF-b ctions noted in Cki- nd SK- RC-5 RCC cells re likely to be consequence of differences in the ctivtion of their downstrem TbR signling pthwys. Our results indicte n lterntive wy of crosstlk between the IGF tyrosine kinse nd TGF-b serine/threonine signling pthwys, in which IGF-I influences phosphoryltion of downstrem signling protein belonging to the TbR signling system. In line with this, in the physiologicl sitution where both IGF-I nd TGF-b re present, the xenogrft Cki- tumors s well s the humn RCC tissues showed strong nucler ccumultion of psmd. Cki- expresses ll members required to trnsduce TGF-b signling: ALK-5, TbRII, Smd, Smd3, nd Smd t protein level (dt not shown). Thus, it is tempting to speculte tht these cells concomitntly express high levels of the generl TbR inhibitory Smd7, rendering these cells insensitive to TGF-b tretment in vitro. IGF-I receptor signling my interfere with Smd7 binding to ALK-5 nd thus mking the Smd phosphoryltion site on the TbR vilble. Further investigtions re required to estblish the moleculr mechnisms by which IGF-I intercts with the TGF-b signling system. Our results demonstrte wys of crosstlk between the IGF nd TGF-b systems in humn RCC cells both t receptor signling s well s protein expression levels. We hve demonstrted tht TGF-b is likely to regulte IGFBP-3 secretion nd to trnsduce some of its cellulr effects in these RCC cells. Moreover, IGF-I enhnces the phosphoryltion of Smd in primry Cki- cells. This enhnced knowledge round the regultion nd functionl roles of growth fctors in RCC, importnt during vrious stges in tumor progression, my be of importnce for future therpeutic pproches. MATERIALS AND METHODS Regents Recombinnt humn IGF-I ws from Phrmci (Stockholm, Sweden). Recombinnt humn IGFBP-3, TGF-b1, nd neutrlizing nti-tgf-b(1 3) mab were from R&D Systems (Abingdon, Oxon, UK). Horserdish peroxidse-linked F(b ) donkey-ntirbbit immunoglobulin ws from Amershm Biosciences (Uppsl, Sweden). The nti-tbrii nd nti-alk-5 were from Snt Cruz Biotechnology Inc. (Snt Cruz, CA, USA). The ffinity-purified rbbit polycolonl Ab nti-psmd, nti-smd, nti-smd3, nd nti-smd, s described previously,,5 were kind gifts from Dr P ten Dijke (Leiden, The Netherlnds). Animls nd in vivo xenogrft tumor model Femle severe-combined immunodeficient C.B-17 mice were bred in-house nd were routinely used t the ge of 1 weeks. Cki- or SK-RC-5 tumor cells (15 1 cells/ml) in. ml phosphtebuffered sline (PBS) with 1% syngenic mouse serum were inoculted intrperitonelly of severe-combined immunodeficient mice. Mice were killed 5 dys fter tumor inocultion, tumors resected, momentrily frozen, nd kept t 71C until further use. Cell lines The humn cler-cell RCC cell line Cki- ws purchsed from Americn Type Culture Collection (Mnsss, VA, USA). The humn cler-cell RCC cell line SK-RC-5 ws gift from Dr SO Wrnr (Centocor, Leiden, The Netherlnds). The cell lines were mintined in R1 medi (Roswell s Prk Memoril Institute medi 1 (BioWhittker, Verviers, Belgium) supplemented with 1% fetl bovine serum (Biotech Line AS, Lynge, Denmrk) nd.1 mg/ ml gentmicin (BioWhittker, Verviers, Belgium)) in humidified 5% CO tmosphere t 371C. All experiments were crried out in serum-free Roswell s Prk Memoril Institute medi 1 with.1 mg/ml gentmicin (SFM). Immunohistochemistry Humn biopsies (provided by Deprtment of Surgery, Lund University Hospitl, Sweden) from RCC (histologiclly clssified 15 Kidney Interntionl () 7,

6 AH Rosendhl nd G Forsberg: IGF-I nd IGFBP-3 in humn RCC originl rticle s four cler cell nd one ppillry crcinom) s well s experimentl xenogrft RCC tumors (four Cki- nd one SK-RC- 5) were dissected nd cryopreserved t 71C until used. Stndrd immunohistochemicl protocol ws used with minor modifictions. Briefly, -mm-thick sections were fixed in % prformldehyde (Sigm, Steinheim, Germny) in PBS (BioWhittker, Verviers, Belgium) for 3 min t room temperture. Endogenous peroxidse ctivity ws quenched for 1 min in drkness by 1% H O (Merck, Drmstdt, Germny) in PBS t room temperture. After blocking with vidin/biotin (Vector SP1, Burlingme, CA, USA) in the presence of 5% norml donkey serum (Snt Cruz Biotechnology Inc., Snt Cruz, CA, USA) for nd min, respectively, t room temperture, sections were incubted with.1 mg/ml nti-psmd, lterntively.3 mg/ml nti-alk-5, nti-smd, nti- Smd3, nti-smd, or mg/ml nti-tbrii in Erle s blnced slt solution-sp buffer (Erle s blnced slt solution (Life Technologies, Prsley, Scotlnd) with.1 M N--hydroxyethylpiperzine-N --ethnesulfonic cid (BioWhittker, Verviers, Belgium) nd.1% Sponin (Riedel-de Hën, Seelze, Germny, ph 7.) t 1C overnight. Following wshing, 1:5 dilution of the horserdish peroxidse-linked donkey-nti-rbbit immunoglobulin in Erle s blnced slt solution-sp, supplemented with % norml donkey serum, ws dded for 1 h t room temperture. The slides were developed for 5 min in diminobenzidine (DAB þ Plus) (Sigm, Steinheim, Germny) nd counterstined with methylgreen (Sigm, Steinheim, Germny). Cell prolifertion ssy Cells were cultured ( 1 3 cells/well Cki- or cells/well SK-RC-5) in SFM in 9-well pltes (Nunc, Roskilde, Denmrk) in the presence or bsence of IGF-I (3 ng/ml) nd/or IGFBP-3 (5 ng/ml) nd vrious concentrtions of TGF-b1 ( 1 ng/ml) or neutrlizing nti-tgf-b(1 3) mab ( mg/ml). Following incubtion for h, cells were pulsed with 3 H-thymidine (NEN, Boston, MA, USA) (.5 mci/well) for h t 371C s mesurement of DNA synthesis nd nlyzed on liquid scintilltion b-counter (LKB WALLAC, Perkin-Elmer Life Sciences, Zventem, Belgium). IGFBP-3 enzyme-linked immunosorbent ssy Cells were cultured ( 1 3 cells/well Cki- or cells/well SK-RC-5) in ml SFM in the presence or bsence of IGF-I (3 ng/ ml), TGF-b1 (5 ng/ml), IGF-I (3 ng/ml) in combintion with TGF-b1 (5 ng/ml), or neutrlizing nti-tgf-b(1 3) Ab ( mg/ml) in 9-well pltes (Nunc, Roskilde, Denmrk). Following incubtion for 7 h, the conditioned medi were collected nd ssyed for IGFBP-3, using IGFBP-3 DuoSet ELISA Development System (R&D Systems, Abingdon, Oxon, UK), ccording to the mnufcturer s instructions. Immunocytochemistry Cells were cultured ( 1 3 cells/well) for h in R1 in eight-well chmber slides (Lb-Tek II Chmber Slide System, Nunc). Cells were then wshed with pre-heted SFM nd subsequently grown for 1 h in SFM with or without the presence of neutrlizing nti-tgf-b(1 3) mab ( mg/ml). Cells were then treted for 1 min with TGF-b1 (5 ng/ml) or 3 min with IGFBP-3 (5 ng/ml), with or without concomitnt IGF-I (3 ng/ml). Cells were subsequently wshed in ice-cold PBS, fixed in % prformldehyde for min t 1C, nd wshed in ice-cold PBS followed by immunohistochemicl stining for psmd, s described in the immunohistochemicl section, with the modifiction tht cells were stined with primry nd secondry Ab for 3 min t 1C. ACKNOWLEDGMENTS We thnk Dr Peter ten Dijke for kindly providing, nd Dr Alexnder Rosendhl for ffinity purifying, the nti-psmd, nti-smd, nti-smd3, nd nti-smd ntibodies. We lso thnk Ms Kristin Behm nd Mr Jn Nilsson for technicl ssistnce with the in vivo work. REFERENCES 1. Gnrr JR, Tory K, Weng Y et l. Muttions of the VHL tumour suppressor gene in renl crcinom. Nt Genet 199; 7: Turner KJ, Moore JW, Jones A et l. Expression of hypoxi-inducible fctors in humn renl cncer: reltionship to ngiogenesis nd to the von Hippel Lindu gene muttion. Cncer Res ; : Dtt K, Nmbudripd R, Pl S et l. Inhibition of insulin-like growth fctor-i-medited cell signling by the von Hippel Lindu gene product in renl cncer. J Biol Chem ; 75: Annth S, Knebelmnn B, Gruning W et l. Trnsforming growth fctor bet1 is trget for the von Hippel Lindu tumor suppressor nd criticl growth fctor for cler cell renl crcinom. Cncer Res 1999; 59: Bserg R, Hongo A, Rubini M et l. 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