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. 2014 Feb 21;9(2):e89114.
doi: 10.1371/journal.pone.0089114. eCollection 2014.

Bone morphogenetic protein signaling protects against cerulein-induced pancreatic fibrosis

Xuxia Gao  1 , Yanna Cao  2 , Dustin A Staloch  1 , Michael A Gonzales  1 , Judith F Aronson  3 , Celia Chao  4 , Mark R Hellmich  4 , Tien C Ko  2
Affiliations

Bone morphogenetic protein signaling protects against cerulein-induced pancreatic fibrosis

Xuxia Gao et al. PLoS One. .

Abstract

Bone morphogenetic proteins (BMPs) have an anti-fibrogenic function in the kidney, lung, and liver. However, their role in chronic pancreatitis (CP) is unknown. The aim of this study was to define the anti-fibrogenic role of BMP signaling in the pancreas in vivo under CP induction. Mice with a deletion of BMP type II receptor (BMPR2(+/-)) were used in this study in comparison with wild-type mice. CP was induced by repetitive cerulein injection intraperitoneally for 4 weeks, and the severity of CP was evaluated. Pancreatic stellate cells (PSCs) were isolated from the mice and treated with BMP2 and TGF-β in vitro, and extracellular matrix protein (ECM) production was measured. Smad and mitogen-activated protein kinase (MAPK) signaling was also evaluated. BMPR2(+/-) mice revealed a greater pancreatic fibrosis, PSC activation and leukocyte infiltration after CP induction compared to wild-type mice (P<0.05). Under CP induction, phospho (p)Smad1/5/8 was elevated in wild-type mice and this effect was abolished in BMPR2(+/-) mice; pSmad2 and pp38(MAPK) were further enhanced in BMPR2(+/-) mice compared to wild-type mice (P<0.05). In vitro, BMP2 inhibited TGF-β-induced ECM protein fibronectin production in wild-type PSCs; this effect was abolished in BMPR2(+/-) PSCs (P<0.05). In BMPR2(+/-) PSCs, pSmad1/5/8 level was barely detectable upon BMP2 stimulation, while pSmad2 level was further enhanced by TGF-β stimulation, compared to wild-type PSCs (P<0.05). BMPR2/Smad1/5/8 signaling plays a protective role against cerulein-induced pancreatic fibrosis by inhibiting Smad2 and p38(MAPK) signaling pathways.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of BMP signaling molecules increased in the pancreas under CP induction.
(A) Representative images of H&E staining. Original magnification×400. (B) mRNA expression of BMP2, BMP4, BMP7, BMPR1a and BMPR2. The mRNA levels were normalized against 18s and quantified as fold of control (CON). *P<0.05 compared with CON. CON mice: n = 4, CP mice: n = 7.
Figure 2
Figure 2. BMPR2 deficiency enhanced pancreatic fibrosis and PSC activation after CP induction.
(A) Representative images of sirius red staining for collagen deposition in the pancreas. (B) Quantification of collagen deposition. CON wild-type: n = 4, CON BMPR2+/−: n = 5, CP wild-type: n = 7, CP BMPR2+/−: n = 9. Original magnification×400. (C) ColIa and FN protein levels in the pancreas by Western blotting. (D) Quantification of ColIa and FN. The protein levels were normalized against GAPDH and quantified as fold of CON wild-type. CON wild-type: n = 4, CON BMPR2+/−: n = 5, CP wild-type: n = 7, CP BMPR2+/−: n = 9. *P<0.05 compared with CON, # P<0.05 compared with CP wild-type. (E) Representative images of immunohistochemistry against α-SMA on paraffin-embedded pancreatic sections. (F) Quantification of α-SMA-positive cells in the periacinar areas. n = 4 mice/group. Original magnification×400. *P<0.05 compared with CON, # P<0.05 compared with CP wild-type.
Figure 3
Figure 3. BMPR2 deficiency increased leukocyte infiltration in the pancreas after CP induction.
(A) Western blotting of pancreatic tissue lysates with anti-CD45 antibody. (B) Quantification of CD45 Western blots. The protein levels were normalized against GAPDH and quantified as fold of CON wild-type. CON wild-type: n = 4, CON BMPR2+/−: n = 5, CP wild-type: n = 7, CP BMPR2+/−: n = 9. *P<0.05 compared with CON, # P<0.05 compared with CP wild-type. (C) Representative images of immunohistochemistry on paraffin-embedded pancreatic sections against CD45. Arrows point to CD45 positive cells. Original magnification×400.
Figure 4
Figure 4. BMPR2 deficiency enhanced pSmad2 level after CP induction.
(A) Western blotting of pancreatic tissue lysates with anti-pSmad1/5/8, -pSmad2, -pSmad3, total-Smad1/5/8, -Smad2 and -Smad3 antibodies. (B) Quantification of the Western blots. The protein levels were normalized against GAPDH and quantified as fold of CON wild-type. CON wild-type: n = 4, CON BMPR2+/−: n = 4–5, CP wild-type: n = 7, CP BMPR2+/−: n = 9. *P<0.05 compared with CON, # P<0.05 compared with CP wild-type.
Figure 5
Figure 5. pp38MAPK signaling increased in BMPR2+/− mice after CP induction.
(A) Western blotting of pancreatic tissue lysates from wild-type and BMPR2+/− mice with anti-pp38MAPK and total p38MAPK antibodies. (B) Quantification of the Western blots. The protein levels were normalized against GAPDH and quantified as fold of CON wild-type. CON wild-type: n = 4, CON BMPR2+/−: n = 5, CP wild-type: n = 7, CP BMPR2+/−: n = 9. *P<0.05 compared with CON, # P<0.05 compared with CP wild-type.
Figure 6
Figure 6. BMPR2 deficiency increased Smad2 phosphorylation in PSCs.
(A) Wild-type and BMPR2+/− PSCs were treated with 250 ng/ml of BMP2 for the indicated time points. The whole cell lysates were subjected to Western blotting with anti-pSmad1/5/8 and total Smad1 antibodies. (B) Quantification of the Western blots. The protein levels were normalized against GAPDH and quantified as fold of 0 min. *P<0.05 compared with 0 min, # P<0.05 compared with the same time points of wild-type. Wild-type: n = 3, BMPR2+/−: n = 3. (C) Wild-type and BMPR2+/− PSCs were treated with 1 ng/ml of TGF-β1 for the indicated time points. Immunofluorescence was performed using antibody against pSmad2 (red). The cell nucleus was stained with DAPI (blue). The images of pSmad2 and pSmad2 merged with DAPI were presented. (D) Quantification of fluorescence intensity from 3 different fields per sample. *P<0.05 compared with 0 min, # P<0.05 compared with the same time points of wild-type PSCs. Original magnification×200.
Figure 7
Figure 7. BMP2/pSmad1/5/8 signaling inhibited TGF-β-induced fibronectin in PSCs.
(A) Wild-type and BMPR2+/− PSCs were pretreated with 250 ng/ml of BMP2 for 30 min followed by 1 ng/ml of TGF-β1 for 48 h. Immunofluorescence was performed using antibody against FN (green), and the cell nucleus was stained with DAPI (blue). The images of FN merged with DAPI were presented. (B) Quantification of fluorescence intensity from 5–10 different fields per slide, *P<0.05 compared with vehicle group, # P<0.05 compared with TGF-β1 group. Original magnification×200.
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