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Figure 2 A VDR-Regulated Transcriptional Network Opposes PSC Activation Show full caption (A) Representative images of primary human CAPSCs treated with vehicle (DMSO) or 100 nM calcipotriol (Cal) for 48 hr and stained with BODIPY 493/503 for detection of neutral lipids. Quantification of percent BODIPY-positive area per cell in three patient samples treated with DMSO or Cal appears below, plotted as the mean + SD. Statistical significance determined by Student’s unpaired t test (∗p < 0.05). Scale bar represents 20 μm. (B) Expression of ACTA2 in 27 primary human CAPSCs treated with vehicle or 100 nM Cal for 48 hr. Values were plotted as DMSO/Cal and normalized to 36B4. (C and D) Heatmap representing selected genes from RNA-seq analysis of primary mouse PSCs treated with DMSO (D) or Cal (C) and harvested on day 3 (preactivated) or day 7 (activated) of culture after isolation (n = 3). VDR target genes Cyp24a1 and Vdr are shown as controls. See also Table S1 . (D) Heatmap showing the relative abundance of negative (top) and positive (bottom) regulators of angiogenesis in activated primary mouse PSCs cultured in the presence of vehicle (DMSO) or Cal. (E) Expression levels of selected genes from the PSC activation or cancer signatures in CAPSCs treated with DMSO or 100 nM Cal for 48 hr. Results are representative of three patient samples and are plotted as the mean + SD qRT-PCR was performed in technical triplicate and values were normalized to 36B4. Statistical significance determined by Student’s unpaired t test (∗p < 0.05). (F) CAPSCs were transfected with siRNA pools against VDR (siVDR) or a non-nontargeting control (siNT). Cells were treated with DMSO or 100 nM Cal for 48 hr and analyzed by qRT-PCR. Values were normalized to 36B4. Results are representative of three patient samples and are plotted as the mean + SD. Statistical significance determined by Student’s unpaired t test (∗p < 0.05; n.s. = not significant). See also Figure S2

Figure S2 VDR Activation Antagonizes the TGFβ/SMAD Pathway in PSCs, Related to Figure 2 Show full caption (A) Primary human CAPSCs treated with vehicle (DMSO, not shown) or 100nM calcipotriol (Cal) for 48h were fixed and stained with BODIPY 493/503 for detection of neutral lipids. Six images (representing 19/27 samples) represent Cal-treated cells and contain cytoplasmic lipid droplets, a hallmark of the quiescent state. (B and C) The hPSC cell line was acutely activated with 1ng/ml TGFβ for 4h, and pretreated with 100nM calcipotriol (Cal) for 16h. Cells were fixed and subject to chromatin immunoprecipitation (ChIP) for SMAD3 and VDR, and rabbit IgG as an isotype control. Chromatin immunoprecipitates were analyzed by QPCR to assess binding of VDR and SMAD3 to the promoter regions of the HAS2 and (C) COL1A1 genes. Rabbit IgG served as an isotype control for both antibodies. Bars indicate mean + SD. ∗p < 0.05 by Student’s t test.

Figure S3 VDR Activation Reduced Inflammation and Fibrosis during Cerulein-Induced Pancreatitis, Related to Figure 3 Show full caption For details of chronic (n = 10) and acute (n = 5) pancreatitis methods, see Extended Experimental Procedures . Pancreata were harvested, sliced, and immediately fixed in formalin or embedded in OCT and frozen. (A) H&E staining of FFPE sections from the indicated treatment groups. Scale bar = 100 μm. (B) Co-immunofluorescence for Collagen I and PSC marker GFAP on frozen sections from the indicated treatment groups. Scale bar = 100 μm. (C) Pancreata from wild-type and Vdr−/− littermates at 6 months of age were harvested and collagen was stained with Sirius Red. Two representative samples are shown per genotype (n = 8). Scale bar = 500 μm.

Figure 3 VDR Ligand Modulates PSC Activation In Vivo Show full caption (A) Expression levels of selected genes in PSCs isolated from mice injected with cerulein (Cer) or cerulein + Cal for 12 weeks (n = 10). Values were normalized to 36b4 and are plotted as the mean + SD. (B) Quantification of immunofluorescent staining for phospho-Stat3 (p-Stat3) on frozen sections from wild-type mice treated with cerulein or cerulein + Cal for 12 weeks (n = 5). (C) Expression levels of selected genes in PSCs isolated from mice injected with Cer or Cer + Cal to induce acute pancreatitis (for details see Extended Experimental Procedures ; n = 5). Values were normalized to 36b4 and are plotted as the mean + SD. (D) Leukocyte recruitment, as measured by CD45-positive cells, in mice with acute pancreatitis (immunofluorescent staining of frozen sections, positive cells in 20× field, n = 5). (E) Fibrosis, as measured by Sirius red staining, in mice with acute pancreatitis (per 20× field, n = 5). (F) Expression levels of selected genes in PSCs isolated from Vdr+/+ and Vdr−/− mice injected with cerulein to induce acute pancreatitis (n = 5). Means + SD are shown; values normalized to B2M. (G) Sirius red-positive area in Vdr+/+ and Vdr−/− mice with acute pancreatitis (per 20× field, n = 5). Statistical significance determined by Student’s unpaired t test (∗p < 0.05). (H) Expression levels of selected genes in PSCs isolated from Vdr+/+ and Vdr−/− mice after treatment with DMSO or 100 nM Cal for 48 hr. Statistical significance determined by Student’s unpaired t test (∗p < 0.05; n.s. = not significant).

These analyses also revealed that PSCs unexpectedly express high levels of the vitamin D receptor (VDR), previously thought not to be expressed in the exocrine pancreas () ( Figures 1 D, 1E, S1 D, and S1E). Importantly, VDR expression is maintained in the cancer-associated PSCs ( Figure 1 F). We focused on this druggable receptor in light of our previous work implicating VDR as a critical regulator of the fibrogenic gene network in closely related hepatic stellate cells () and due to the established anti-inflammatory actions of 1,25(OH)and its analogs (). Here, we used calcipotriol (Cal), a potent and nonhypercalcemic vitamin D analog to control VDR induction (). While not present in any postsurgical CAPSCs, surprisingly, Cal treatment induced lipid droplet formation in 19/27 primary patient samples ( Figures 2 A and S2 A) and decreased expression of αSMA (ACTA2) in 24/27 patient samples ( Figure 2 B). This strongly supports the idea that the activation state is controllable in a signal-dependent fashion. To assess the genome-wide effects of VDR activation in PSCs, we performed transcriptome analysis of preactivated and activated PSCs grown in the presence or absence of VDR ligand. While Cal treatment affected gene expression in preactivated PSCs (significantly increased and decreased expression of 307 and 431 genes, respectively), VDR activation had a more widespread transcriptional response in activated PSCs (664 and 1,616 genes with significantly increased and decreased expression, respectively). Notably, we observed a Cal-dependent inhibition of the activation and cancer signatures in PSCs ( Figure 2 C; Table S1 ), including suppression of negative regulators of angiogenesis such as Thbs1 and induction of positive regulators of angiogenesis like Mmp9 () ( Figure 2 D). Similar effects of Cal treatment were observed on selected candidate genes in human CAPSCs ( Figure 2 E). Furthermore, these effects were dependent on VDR, as small interfering RNA (siRNA)-mediated knockdown of the receptor abrogated Cal-induced expression changes ( Figure 2 F). To explain, in part, the broad impact of VDR on the PSC activation program, we assessed genomic crosstalk between VDR and the TGF-β/SMAD pathway () that we previously demonstrated in hepatic stellate cells (). Consistent with an inhibitory effect on TGF-β/SMAD signaling, Cal increased VDR binding while decreasing SMAD3 binding in the promoter regions of fibrogenic genes ( Figures S2 B and S2C). To determine whether VDR activation decreased PSC activation in vivo, we induced experimental chronic pancreatitis in wild-type mice using the cholecystokinin analog cerulein () and coadministered Cal throughout disease progression. Compared to mice receiving cerulein alone, Cal-treated animals displayed attenuated inflammation and fibrosis, consistent with decreased PSC activation ( Figures S3 A and S3B). Expression of activation and cancer signature genes was decreased in isolated PSCs from mice treated with Cal compared to controls ( Figure 3 A). Reductions were observed on activation signature genes that are of functional significance in the tumor microenvironment, including ECM components, inflammatory cytokines, and growth factors. In addition, Acta2 expression, which is associated with cell motility, trended downward. Further, reduced induction of phospho-Stat3 was observed in Cal-treated mice ( Figure 3 B), consistent with decreased inflammatory signaling from the stroma. Notably, Stat3 activation has been established as a mechanistic link between inflammatory damage and initiation of PDA (). Cal treatment during acute pancreatitis in wild-type mice similarly impaired activation-associated changes in PSC gene expression ( Figure 3 C) and reduced leukocyte infiltration and fibrosis ( Figures 3 D and 3E). Strikingly, pancreata from Vdrmice displayed spontaneous periacinar and periductal fibrosis ( Figure S3 C), further supporting a role for VDR in opposing PSC activation. Consistent with this notion, activation-associated changes in PSC gene expression were augmented in cerulein-induced acute pancreatitis in Vdrmice ( Figure 3 F) and were accompanied by increased fibrosis ( Figure 3 G). Furthermore, Cal treatment of culture-activated PSCs from Vdrmice demonstrated the VDR dependence of the observed gene expression changes ( Figure 3 H). Together, these results suggest that VDR acts as a master genomic regulator of the PSC activation program, and VDR induction by ligand promotes the quiescent PSC state both in vitro and in vivo.