Activated effects of parathyroid hormone-related protein on human hepatic stellate cells

Abstract

Background & aims: After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP) has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-β1), which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC) and LX-2 cell lines.

Methods: TGF-β1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR) after HSCs or LX-2 cells were treated with PTHrP(1-36) or TGF-β1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-β1 secretion was measured with enzyme-linked immunosorbent assay (ELISA) of HSC cell culture media.

Results: In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-β1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-β1 secretion in cultured media from HSCs.

Conclusions: PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-β1, highlighting the potential effects of PTHrP in the liver.

Figure 1. Alpha -SMA immunofluorescence.

Alpha –SMA was detected in HSCs treated with 100 nM PTHrP(1–36) for 48 h (A), and stronger expression was found in LX-2 cells (B). Cells were counterstained with DAPI to identify nuclei.

Figure 2. Increased production of α-SMA in HSC and LX-2 cells after PTHrP(1–36) treatment.

After treatment with 100(1–36) for 48 h, the α-SMA protein (43 kd) levels assessed by western blot (A) and mRNA levels measured by q-PCR were increased in HSCs (B). The same patterns were observed in LX-2 (C and D). * p<0.05 compared to the control group.

Figure 3. Increased production of collagen I in HSC and LX-2 cells after treatment with PTHrP(1–36).

After treatment with PTHrP(1–36) (100 nM) for 48 h, collagen I (138 kd) protein levels were assessed by western-blot (A); mRNA expression was measured with q-PCR and found to be increased in both HSCs (B), and LX-2 cells (C and D). * p<0.05 compared to the control group.

Figure 4. Collagen I expression in HSCs and LX-2 cells after TGF-β1 and PTHrP treatment.

After treatment with TGF-β1 (1 ng/ml) alone or with PTHrP (100 nM) for 48 h, collagen I (138 kd) total protein was assessed by western blot (A); mRNA expression was measured with q-PCR and found to be increased in both HSCs (B), and LX-2 cells (C and D). * p<0.05 compared to the control group. No significant difference was observed between treated groups.

Figure 5. Increased production of MMP-2 in HSCs and LX-2 cells after PTHrP(1–36) treatment.

After a 48(1–36), the MMP-2 (72 kd) total protein levels were assessed by western blot (A); mRNA expression was measured with q-PCR and found to be increased in HSC (B). Similar patterns were observed for LX-2 cells (C and D). * p<0.05 compared to the control group.

Figure 6. PTHrP(1–36) induced TGF-β1 in HSC and LX-2 cells.

After a 48(1–36), TGF -β1 (45 kd) total protein levels were assessed by western-blot (A); mRNA expression was measured with q-PCR and found to be increased in HSC (B), and LX-2 (C and D). * p<0.05 compared to the control group.

Figure 7. TGF-β1 secretion by HSC after treatment with PTHrP(1–36).

Total TGF -β1 secretion in cell culture medium after treatment with 100 nM PTHrP(1–36) for 48 h. * p<0.05 compared to the control group.