Gremlin is a key pro-fibrogenic factor in chronic pancreatitis

Abstract

The current study aims to identify the pro-fibrogenic role of Gremlin, an endogenous antagonist of bone morphogenetic proteins (BMPs) in chronic pancreatitis (CP). CP is a highly debilitating disease characterized by progressive pancreatic inflammation and fibrosis that ultimately leads to exocrine and endocrine dysfunction. While transforming growth factor (TGF)-β is a known key pro-fibrogenic factor in CP, the TGF-β superfamily member BMPs exert an anti-fibrogenic function in CP as reported by our group recently. To investigate how BMP signaling is regulated in CP by BMP antagonists, the mouse CP model induced by cerulein was used. During CP induction, TGF-β1 messenger RNA (mRNA) increased 156-fold in 2 weeks, a BMP antagonist Gremlin 1 (Grem1) mRNA levels increased 145-fold at 3 weeks, and increases in Grem1 protein levels correlated with increases in collagen deposition. Increased Grem1 was also observed in human CP pancreata compared to normal. Grem1 knockout in Grem1 (+/-) mice revealed a 33.2 % reduction in pancreatic fibrosis in CP compared to wild-type littermates. In vitro in isolated pancreatic stellate cells, TGF-β induced Grem1 expression. Addition of the recombinant mouse Grem1 protein blocked BMP2-induced Smad1/5 phosphorylation and abolished BMP2's suppression effects on TGF-β-induced collagen expression. Evidences presented herein demonstrate that Grem1, induced by TGF-β, is pro-fibrogenic by antagonizing BMP activity in CP.

Key messages: • Gremlin is upregulated in human chronic pancreatitis and a mouse CP model in vivo. • Deficiency of Grem1 in mice attenuates pancreatic fibrosis under CP induction in vivo. • TGF-β induces Gremlin mRNA and protein expression in pancreatic stellate cells in vitro. • Gremlin blocks BMP2 signaling and function in pancreatic stellate cells in vitro. • This study discloses a pro-fibrogenic role of Gremlin by antagonizing BMP activity in chronic pancreatitis.

Keywords: Bone morphogenetic protein antagonists; Cerulein; Gremlin; Pancreatic fibrosis; Pancreatic stellate cells.

Figure 1. CP increases expression of pancreatic TGF-β1 and BMP antagonists

Pancreata were harvested from C57BL/6 mice following 1, 2, or 3 weeks CP induction with cerulein or after 5 weeks recovery following 3 weeks CP induction (3w+5w recovery). Total RNAs were extracted and converted to cDNAs. mRNA expression levels of TGF-β1, Grem1, Noggin, and Chordin1 were measured by qPCR. The specific signals were normalized against 18S rRNA and expressed as fold of respective 0 week expression levels (mean±SEM). n=5-9/group. *P<0.05 compared with respective 0 week using one-way ANOVA and multiple comparison test (Dunn's method).

Figure 2. Collagen deposition increases in mouse pancreata in a time-dependent manner following CP induction

Pancreata were harvested from Swiss Webster mice following up to 8-week CP induction with cerulein or saline control (CON). A. Images of Sirius red staining for collagen deposition. Original magnification= 400X. B. Quantification. Data are expressed as fold of CON, and presented as mean±SEM. n=4-6 mice/group. *P<0.05 compared to time-matched controls using Student's t-test.

Figure 3. Grem1 expression increases in mouse pancreata in a time-dependent manner following CP induction

Pancreata were harvested from Swiss Webster mice following up to 8-week CP induction with cerulein or saline control (CON). A. Images of immunohistochemistry using a goat anti-Grem1 antibody. IHC CON: Goat IgG isotype control. B. Quantification of Grem1 IHC. C. Images of Western blots. Pos: recombinant mouse Grem1 protein as a positive control. D. Quantification of Western blots. Grem1 levels are normalized against GAPDH, expressed as fold of CON, and presented as mean±SEM. n=4-6 mice/group. *P<0.05 compared to time-matched controls using Student's t-test.

Figure 4. Grem1 expression increases in the pancreata of human CP

A. Paraffin sections from normal and CP human pancreatic tissue underwent immunohistochemical staining using a goat anti-Grem1 (arrow) antibody and a rabbit anti-pSmad1/5 (arrow head) antibody. IHC CON1: Goat IgG isotype control. IHC CON2: Rabbit IgG isotype control. B. Histoscores of Grem1 and pSmad1/5 were assigned to each of 5 different fields/slide as follows: 0 for no staining; 1+ for <30% cells positively stained; 2+ for 30-60% cells positively stained; and 3+ for >60% cells positively stained. Original magnification= 400X. Mean histoscores±SEM are presented for each group. n=4 normal pancreata; n=7 CP. *P<0.05 compared to normal pancreata using Student's t-test.

Figure 5. Grem1 haplodeficiency in mice attenuates cerulein-induced pancreatic fibrosis

Representative images are shown for Sirius red staining of control (CON) and CP pancreatic paraffin sections from Wild-type (WT) and Grem1^+/− mice following 4-week CP induction with cerulein. Original magnification= 400X. Quantification of collagen area is expressed as a fold of WT control±SEM. n=6 mice/group. *P<0.05 compared to WT control, #P<0.05 compared to WT CP using one-way ANOVA and multiple comparison test (Holm-Sidak method).

Figure 6. TGF-β induces Grem1 in mPSCs

mPSCs were treated with either vehicle (Veh) or TGF-β1 (1 ng/ml) for 24 h for qPCR and for 48 h for IF and Western blotting. A. Grem1 mRNA levels measured by qPCR, expressed as fold of Veh, and presented as mean±SEM from 3 independent sets of treatments. B. Images and quantification of Western blots. Grem1 levels are normalized against GAPDH, expressed as fold of Veh, and presented as mean±SEM. C. Western blots probed with Grem1 antibody or neutralized Grem1 antibody. +T: mPSCs treated with TGF-β1 for 48 h. rGrem1: recombinant mouse Grem1 protein as a positive control. D. Representative merged images of IF using antibodies against Grem1 (green) with DAPI nuclear staining (blue). Quantification of IF is expressed as fold of Veh and presented as mean±SEM. n=5-10 independent fields/slide. Original magnification= 200X. *P<0.05 compared to Veh using Student's t-test.

Figure 7. Grem1 blocks BMP2 signaling and anti-fibrogenic effects in mPSCs

A. Representative Western blot images and quantification of Grem1 dose curve study. mPSCs were pretreated with Grem1 (0, 50, 500 ng/ml) for 30 min followed by BMP2 treatment (50 ng/ml) for 30 min. B. Representative Western blot images and quantification. mPSCs were treated with Veh, TGF-β1 (1 ng/ml), BMP2 (50 ng/ml) for 30 min followed by TGF-β (1 ng/ml); or Grem1 (500 ng/ml) for 30 min followed by BMP2 (50 ng/ml) for 30 min and then TGF-β1 (1 ng/ml) for 30 min. pSmad1/5 levels are normalized against GAPDH and expressed as fold of Veh. C. The cells were treated as indicated for 24 h. Col1a1 mRNA levels were measured by qPCR. D. The cells were treated as indicated for 48 h. Col1a1 protein levels were measured by IF. Data are presented as mean±SEM. n=3. *P<0.05 compared to Veh, #P<0.05 compared to BMP2 or TGF-β treatment alone using one-way ANOVA and multiple comparison test (Holm-Sidak method).