Adrenocortical Stem and Progenitor cells: Growth factors, signaling and transcription factors. Implications for adrenal diseases. Gary D.

Transcription

1 Adrenocortical Stem and Progenitor cells: Growth factors, signaling and transcription factors Implications for adrenal diseases Gary D. Hammer Condensation of the Ventral Intermediate Mesoderm Forms the Urogenital Ridge Mesonephros (embryonic kidney) Coelomic epithelium Adrenogonadal primordium Coelomic Epithelium Species: Mouse Day Gestation: Approx. Human Age: 5-6 weeks View: Transverse Cut 1

2 SF-1 Expression Specifically Marks Developing Steroidogenic Tissues Mesonephros (embryonic kidney) Adrenogonadal primordium Nathan Bingham and Keith Parker Shared SF-1 lineage of adrenal & gonads 2

3 Adrenocortical development Lineage relationship and signaling between 3 structures fetal zone mesenchymal capsule definitive zone definitive zone Adrenocortical development 3

4 Adrenocortical development Defining a fetal zone specific enhancer of SF-1: FAdE Zubair et al. MCB 26: (2006) 4

5 Fetal zone cells give rise to definitive zone cells Zubair et al. MCB 28: (2008) Restricted window in which the FAdE active fetal zone cells can develop into adult adrenal cortex. 5

6 SF-1 expression (FAdE) is shut off and a putative DAdE is activated in fetal zone cells that then become definitive zone cells Does an SF-1 negative state serve as an intermediate during the FadE to DadE switch 6

7 Cells of the SF-1 negative capsule are also derived from the fetal zone cells Mohammed Zubair Do fetal zone cells give rise to 2 independent cell populations? (SF-1 negative capsule and SF-1 positive definitive zone cells)? OR 7

8 Does an SF-1 negative state serve as an intermediate during the FadE to DadE switch? (SF-1 negative capsule give rise to the definitive zone cells)? Emergence of SF-1 positive cells within clusters of SF-1 negative capsular/subcapsular spindle cells SF-1 1 negative capsular/subcapsular subcapsular spindle cells Capsular cells are SF-1 1 negative Cortical cells are defined by SF-1 SF-1 1 positive cortical cells Bielinska et al Endocrinology 144( 9): ,

9 Upstream stimulatory factors (USF1/2) regulates basal SF-1 expression (+1) -123 E box CAAT box GA rich element bhlh -123 E box (+1) USF -123 E box (+1) Harris and Mellon (1998) Mol. Endocrinology 12: POD1: Inhibitor of SF-1 mediated differentiation POD1 -HLH transcription factor -inhibits differentiation -inhibits SF-1 in adrenal cells -localizes to adrenal capsule POD1 USF1 Ebox SF-1 promoter Lu et al, Mech of Devel 73: 23-32,

10 Pod-1(Capsulin, TCF21) is restricted to SF-1 negative capsular cells (Loss of Pod-1 induces SF-1 expression in capsule) 2.0 SF-1 expression Pod1 -/- Pod1 +/- Claudimara Lofti in collaboration with Sue Quaggin Adrenocortical homeostasis - Capsular niche Sf1 positive fetal zone cells (precursor cells) give rise to Sf1 negative capsular stem cells that serve to replenish definitive zone subcapsular progenitor cells 10

11 Adrenocortical homeostasis - Capsular niche Sf1 positive fetal zone cells (precursor cells) give rise to Sf1 negative capsular stem cells that serve to replenish definitive zone subcapsular progenitor cells Adrenocortical homeostasis - Capsular niche Sf1 positive fetal zone cells (precursor cells) give rise to Sf1 negative capsular stem cells that serve to replenish definitive zone subcapsular progenitor cells 11

12 Adrenocortical homeostasis - differentiation of definitive cortex Sf1 positive differentiated definitive cells Adrenocortical homeostasis Capsular niche: stem cells gives rise to subcapsular progenitor cells of definitive cortex Subcapsular progenitors: undifferentiated cells give rise to differentiated cortical cells Understanding how signaling pathways control stem/progenitor cells is critical to our quest to understand and treat adrenal diseases 12

13 subcapsular progenitor cells: transiently amplifying cells Capsule Proliferating cells zg Cortex zf Medulla PCNA zr Brendan Looyenga subcapsular progenitor cells: zona Undifferentiated/Intermedia of rat Mitani et al, Biochimica et Biophysica Acta (3): , 324,

14 Capsular/subcapsular implants proliferate and differentiate into functional adrenal tissue H&E SF-1 IHC Felix Beuschlein Adrenal hypoplasia and cancer syndromes: Stem cell diseases? stem cell signaling defects associated with adrenal disease stem cell self renewal Wnt signaling/dax1 (adenomatous polyposis coli syndrome/cahypoplasia) stem cell fate TGF signaling (pediatric low grade ACC fetal blastoma) stem cell niche/fate IGF signaling (Beckwith-Wiedemann syndrome ) stem cell apoptosis p53 (Li-Fraumeni syndrome) Understanding how these and other signaling pathways control tissue stem cells is critical to our quest to understand and treat adrenal growth disorders Goal: Identify new genes that can be TARGETS for new therapies Wnt antagonists IGF antagonists 14

15 Binary model of PARACRINE factor regulation of adrenal capsule/subcapsule fate Alex Kim and Isabella Finco PARACRINE factor regulation of transcriptional cascades determine self-renewal (proliferation) and cell fate (differentiation) self renewal paracrine activation IGF Wnt transcription activation Dax-1 Sf-1 SHH fate 15

16 Wnt signaling Capsule as niche Wnt signaling gatekeeper of the non-committed fate of subcapsular progenitor cells progenitor cell proliferative non-steroidogenic Wnt responsive progenitor differentiated cortical cells non-proliferative steroidogenic Wnt non-responsive differentiated cell Keith Parker 16

17 Canonical Wnt signaling emerges under capsule in definitive cortex becoming restricted to subcapsular cells Wnt 2b Wnt 4 adr Dev. Dyn. 222: 26 Endo 143: 4358 Alex Kim Adrenal-restricted ablation of Wnt Signaling X SF-1 CRE partial (recombination in some cells) floxed βcat SF-1 CRE complete (recombination in all cells) 17

18 Newborn complete -catenin null mice exhibit absence of adrenals Complete -catenin null mice exhibit cessation of definitive cortex formation SF-1 WT β-cat SF-1 TH KO β-cat 18

19 Newborn partial -catenin null mice exhibit normal adrenals Aging partial -catenin null mice exhibits late adrenocortical cell loss 19

20 Adrenal stem/progenitor cell depletion: A common mechanism of adrenal failure? β-catenin null mouse IMAGe syndrome Tan et al., Am J Med Genet 2006 normal (mouse) acd mouse AHC, DAX1(NR0B1) mutation Lack & Kozakewich Pathology ofthe Adrenal Gland, 1990 Dax-1 as downstream mediator of Wnt signaling gatekeeper of multipotency of subcapsular progenitor cells progenitor cell proliferative non-steroidogenic Dax-1 positive progenitor differentiated cortical cells non-proliferative steroidogenic Dax-1 negative differentiated cell Larry Jameson 20

21 Dax-1 -paradox Nuclear receptor cloned as gene responsible for X-linked Congenital Adrenal Hypoplasia Dax-1 in vivo Human Loss of Function: Adrenal HYPO function Mouse Loss of Function: Adrenal HYPERfunction Dax-1 in vitro SF-1 CCAAGGTCA Dax-1 SF-1 Dax-1 CCAAGGTCA ta rg e t gene or Young Dax-1 null mice exhibit enhanced ACTH responsiveness 600 Corticosterone (ng/ml) Dax-1 null Wild type minutes Joshua Scheys 21

22 Young Dax-1 null mice exhibit enhanced ACTH responsiveness relative ACTH/CS ratio WT Dax1 null Age (weeks) Young Dax-1 null mice exhibit enhanced ACTH responsiveness BUT Old Dax-1 mice exhibit loss of ACTH responsiveness relative ACTH/CS ratio WT Dax1 null Age (weeks) 22

23 Aging Dax-1 null mice exhibit decreased StAR expression (despite early enhanced steroidogenesis) Relative StAR expression Wild type Dax-1 null Weeks of age Aging Dax-1 null mice exhibit decreased SF-1 expression (perhaps reflective of loss of early subcapsular proliferation) Relative SF-1 expression Wild type Dax-1 null Weeks of age 23

24 Enhanced adrenal proliferation in young Dax-1 null mice is lost over time 20 BrdU/Adrenal Dax1 KO 5 Wt Age (weeks) Aging Dax-1 null mice exhibit features of cytomegalic congenital adrenal hypoplasia (Hyperfunction + variable temporal onset of adrenal failure in pts with Dax-1 mutations) Dax-1 null Wild type stochastic depletion of progenitor cells 24

25 Dax1 expression is enriched in the subcapsular adrenal cortex LacZ Wnt-Gal β-catenin SF-1 Dax-1 β-cat SF-1 Dax-1 Physiologic regulation of Dax-1 Wnt (paracrine - capsule) β-cat SF-1 GR Dax-1 glucocorticoid (endocrine adrenal) Brian Gummow 25

26 Physiologic regulation of Dax-1 Wnt (paracrine - capsule) β-cat SF-1 GR Dax-1 ACTH (endocrine - pituitary) glucocorticoid (endocrine adrenal) Brian Gummow Adrenal-restricted constitutive activation of Wnt Signaling X floxed APC SF-1 CRE 26

27 Young APC null mice exhibit accumulation of β-catenin/dax-1 positive cells at medullary boundary (expansion of undifferentiated progenitors?) WT Apc low KO 9 Weeks 15 Weeks 30 Weeks ABC WT PCNA KO β-actin Young APC null mice exhibit activation of β-catenin target genes commensurate with down-regulation of differentiation genes (expansion of undifferentiated progenitors?)

28 Adrenal-specific KO of APC exhibits increase in adrenal size over time Adrenal weight (mg) KO WT Age (Weeks) Aging APC null mice develop adrenocortical adenomas (dysplasia - adenoma progression?) WT 15 Weeks 30 Weeks 45 Weeks H&E (4x) H&E (10x) Sf1 β-catenin 28

29 Aging APC mice develop adrenocortical carcinoma (adenoma carcinoma progression?) adrenal tumor APC KO adrenal wild type kidney size (mm) Wnt/β-catenin status marks variation in gene expression in human ACC Familial ACC APC Sporadic ACC - β-catenin Tom Giordano 29

30 Wnt/β-catenin activation in ACC predicts worse survival Survival Probability Membranous Nuclear β-catenin membranous β-catenin nuclear years Goal: Utilize upcoming Wnt/β-catenin inhibitors in ACC Wnt/β-catenin status marks variation in gene expression in ACC BUT NOT adrenal adenomas (ACA) -Wnt -Wnt +Wnt -Wnt +Wnt normal ACA ACC 30

31 IGF signaling as candidate that synergizes with Wnt/ -catenin in ACC Beckwith-Wiedemann Syndrome: Overgrowth syndrome with elevated risk of embryonic tumors (Wilms Tumor, ACC and others) 80% of cases associated with chromosome 11 defect Loss Of Imprinting or (LOI) Loss Of Heterozygosity (LOH) of 11p15.5 = HIGH IGF2 LOI of IGFII locus in stem cells as initiation event in cancer IGF2 is the most up-regulated gene in adrenal cancer (and p57 and H19 are coordinately down-regulated) normal ACA ACC H19 IGF2 p57 31

32 ZAC1 is expressed in stem cells and controls imprinting of IGFII locus Jelininc, P. and Shaw, P. J Pathol 2007; 211: ZAC1 is downregulated in pediatric ACC West, A. N. et al. Cancer Res 2007;67: ZAC1 is expressed in capsular/subcapsular cells IGF2 p57 ZAC1 mouse e14.5 Jelininc, P. and Shaw, P. J Pathol 2007; 211:

33 Cross talk between Wnt and IGF signaling in adrenal stem cell niche IGF Wnt Adrenal IGF2 LOI (constitutive IGF2) Adrenal APC null (constitutive Wnt) Ferdous Barlaskar Aging APC null mice exhibit increased adrenal IGF2 and decreased p57kip2 (only in ACC of aging APC null mice) (not in ACA of young APC null mice) 2 Fold Expression Igf2 p57kip2 0 33

34 IGF2 LOI mice exhibit commensurate increase in IGF2 Relative IGF2 expression 2 1 wt IGF2 LOI Cre- Cre low IGF2 LOI mice exhibit increased β-catenin staining in subcapsular area Cre high β-catenin expression Total Akt p-akt Ser473 wt Total β-catenin Active β-catenin H19 β-actin 34

35 Cross talk between Wnt and IGF signaling Jin, T. et al Cellular Signalling 20(10): PARACRINE factor regulation of transcriptional cascades determine self-renewal (proliferation) and cell fate (differentiation) self renewal paracrine activation IGF Wnt transcription activation Dax-1 Sf-1 SHH fate 35

36 Understanding how signaling pathways control stem/progenitor cells is critical to our quest to understand and treat adrenal diseases 36