Progesterone stimulates the proliferation of female and male cholangiocytes via autocrine/paracrine mechanisms - PubMed (original) (raw)

. 2008 Jul;295(1):G124-G136.

doi: 10.1152/ajpgi.00536.2007. Epub 2008 May 29.

Sharon DeMorrow, Heather Francis, Yoshiyuki Ueno, Eugenio Gaudio, Shelley Vaculin, Julie Venter, Antonio Franchitto, Paolo Onori, Bradley Vaculin, Marco Marzioni, Candace Wise, Metaneeya Pilanthananond, Jennifer Savage, Lisa Pierce, Romina Mancinelli, Gianfranco Alpini

Affiliations

Progesterone stimulates the proliferation of female and male cholangiocytes via autocrine/paracrine mechanisms

Shannon Glaser et al. Am J Physiol Gastrointest Liver Physiol. 2008 Jul.

Retraction in

Abstract

During cholestatic liver diseases, cholangiocytes express neuroendocrine phenotypes and respond to a number of hormones and neuropeptides by paracrine and autocrine mechanisms. We examined whether the neuroendocrine hormone progesterone is produced by and targeted to cholangiocytes, thereby regulating biliary proliferation during cholestasis. Nuclear (PR-A and PR-B) and membrane (PRGMC1, PRGMC2, and mPRalpha) progesterone receptor expression was evaluated in liver sections and cholangiocytes from normal and bile duct ligation (BDL) rats, and NRC cells (normal rat cholangiocyte line). In vivo, normal rats were chronically treated with progesterone for 1 wk, or immediately after BDL, rats were treated with a neutralizing progesterone antibody for 1 wk. Cholangiocyte growth was measured by evaluating the number of bile ducts in liver sections. The expression of the progesterone synthesis pathway was evaluated in liver sections, cholangiocytes and NRC. Progesterone secretion was evaluated in supernatants from normal and BDL cholangiocytes and NRC. In vitro, NRC were stimulated with progesterone and cholangiocyte supernatants in the presence or absence of antiprogesterone antibody. Aminoglutethimide was used to block progesterone synthesis. Cholangiocytes and NRC express the PR-B nuclear receptor and PRGMC1, PRGMC2, and mPRalpha. In vivo, progesterone increased the number of bile ducts of normal rats, whereas antiprogesterone antibody inhibited cholangiocyte growth stimulated by BDL. Normal and BDL cholangiocytes expressed the biosynthetic pathway for and secrete progesterone. In vitro, 1) progesterone increased NRC proliferation; 2) cholangiocyte supernatants increased NRC proliferation, which was partially inhibited by preincubation with antiprogesterone; and 3) inhibition of progesterone steroidogenesis prevented NRC proliferation. In conclusion, progesterone may be an important autocrine/paracrine regulator of cholangiocyte proliferation.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

A: immunofluorescence for progesterone receptors (PR, PGRMC1, and PGRMC2) in liver sections from normal and bile duct-ligated (BDL) female and male rats demonstrates that bile ducts express these receptors (red staining). Colocalization with CK-19 (green staining, a cholangiocyte-specific marker) of the bile ducts expressing the progesterone receptor is also visible. Bar = 20 μm. B: immunohistochemistry for progesterone receptors (PR, PGRMC1, and PGRMC2) in liver secretion from normal and BDL female male rats confirms receptor expression by bile ducts. Low-level expression of the progesterone receptor was expected in hepatocytes (36). C: by immunofluorescence, progesterone receptors (PR, PGRMC1, and PGRMC2) were expressed by normal rat intrahepatic cholangiocyte cultures (NRC). Specific immunoreactivity is shown in green and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). No staining was visible when primary antibodies were omitted. Bar = 50 μm. D: RT-PCR in pure cholangiocytes from normal and BDL female and male rats and NRC shows that cholangiocytes express the PR-A/B and PGRMC1, PGRMC2, and mPRα. The expression of GAPDH, the housekeeping gene, was similarly expressed by freshly isolated cholangiocytes and NRC. E: by immunoblotting, cholangiocytes from normal and BDL female and male rats and NRC express the PR-B (but not PR-A). The expression of β-actin, the housekeeping gene, was similar in freshly isolated cholangiocytes and NRC. PR, progesterone receptor; PGRM, progesterone receptor membrane component protein.

Fig. 2.

Fig. 2.

Measurement of the number of cytokeratin-19 (CK-19)-positive cholangiocytes in liver sections from the selected groups of animals. A: female rats. B: male rats. Administration of progesterone to normal female or male rats increased the number of CK-19-positive cholangiocytes compared with the corresponding normal female or male rats treated with saline. The administration of the nuclear progesterone receptor antagonist RU-486 to normal female or male rats prevented progesterone-induced increase in cholangiocyte growth. In addition, administration of antiprogesterone antibody to female or male BDL rats (immediately after BDL) decreased the number of CK-19-positive cholangiocytes compared with liver sections from the corresponding female or male BDL rats treated with nonimmune serum. Original magnification, ×10. Data are means ± SE of 5 values obtained from the 3 slides evaluated per each group of animal.

Fig. 3.

Fig. 3.

A: immunofluorescence for key proteins in the progesterone steroidogenesis pathway [steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (p450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD)] in liver sections from normal and BDL female and male rats demonstrates that bile ducts express these steroidogenesis pathway proteins (red staining). Colocalization with CK-19 (green staining, a cholangiocyte-specific marker) of the bile ducts expressing StAR, p450scc, and 3β-HSD is also visible. Bar = 20 μm. B: immunohistochemistry in liver sections from normal and BDL female and male rats for StAR, p450scc, and 3β-HSD confirms bile duct expression, with expression of some components in hepatocytes. C: by immunofluorescence, NRC expressed StAR, p450scc, and 3β-HSD, key proteins in the progesterone steroidogenesis pathway. Specific immunoreactivity is shown in green and nuclei were stained with DAPI (blue). No staining was visible when primary antibodies were omitted. Bar = 50 μm. D: RT-PCR in pure cholangiocytes from normal and BDL female and male rats and NRC shows that cholangiocytes express mRNA messages for StAR, p450scc, and 3β-HSD.

Fig. 4.

Fig. 4.

Progesterone levels in cholangiocyte supernatants and serum from normal and BDL female and male rats. A: progesterone levels in normal female cholangiocyte supernatants were significantly higher than those of normal male rats. Following BDL, there is a significant decrease in progesterone in the supernatants of both female and male rat cholangiocytes compare to normal cholangiocytes. Data are means ± SE of 5 experiments. *P < 0.05 normal female vs. normal male. #P < 0.05 normal female vs. BDL female. &P < 0.05 normal male vs. BDL male. B and C: serum progesterone levels were significantly higher in normal female rats compared with normal male rats. BDL had no significant effects on serum progesterone levels. Chronic administration of progesterone to normal male and female rats significantly elevated serum progesterone levels. Administration of antiprogesterone significantly reduced serum progesterone levels in both BDL female and male rats. Data are means ± SE of 5 experiments. *P < 0.05 normal vs. normal + progesterone. #P < 0.05 BDL vs. BDL + antiprogesterone.

Fig. 5.

Fig. 5.

Effect of normal and BDL cholangiocyte supernatant (in the absence or presence of antiprogesterone antibody) on NRC proliferation. A: consistent with the concept that cholangiocytes regulate their own growth by an autocrine mechanism, the supernatant from primary cultures of normal female and male cholangiocytes stimulated the proliferation of NRC compared with basal unstimulated NRC. In vitro pretreatment of NRC with antiprogesterone antibody, prior to stimulation with cholangiocyte supernatant, partly inhibited progesterone-stimulated cholangiocyte proliferation. Data are means ± SE of 5 experiments. B: in a similar fashion, the supernatant from primary cultures of BDL female and male cholangiocytes also stimulated the proliferation of NRC compared with basal unstimulated NRC (although to a smaller degree than normal supernatant). The proliferation was also partly inhibited by antiprogesterone. Data are means ± SE of 5 experiments. *P < 0.05 vs. corresponding basal value. #P < 0.05 vs. NRC treated with supernatant from normal (or BDL) female cholangiocytes. &P < 0.05 vs. NRC treated with supernatant from normal male (or BDL) cholangiocytes.

Fig. 6.

Fig. 6.

Dose-dependent effects of progesterone (10−10 to 10−6 M for 24 h) on NRC proliferation. Progesterone stimulates a dose-dependent increase in NRC proliferation. Data are means ± SE of 5 experiments. *P < 0.05 vs. basal.

Fig. 7.

Fig. 7.

Effect of aminoglutethimide (AMG) inhibition of cholangiocyte steroidogenesis on NRC proliferation. Inhibition of progesterone steroidogenesis by in vitro treatment of NRC with AMG prevented cholangiocyte proliferation evaluated by immunoblots for PCNA. *P < 0.05 vs. basal. Data are means ± SE of 4 experiments. *P < 0.05 vs. basal.

References

    1. Alpini G, Glaser S, Ueno Y, Pham L, Podila PV, Caligiuri A, LeSage G, LaRusso NF. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 274: G767–G775, 1998. - PubMed
    1. Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 81: 569–578, 1988. - PMC - PubMed
    1. Alpini G, Phinizy JL, Glaser S, Francis H, Benedetti A, Marucci L, LeSage G. Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 284: G1066–G1073, 2003. - PubMed
    1. Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary epithelia. In: The Liver: Biology and Pathobiology (4th ed.), edited by Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby W, Schachter D, and Shafritz DA. Philadelphia, PA: Lippincott Williams & Wilkins, 2001, p. 421–435.
    1. Alpini G, Ueno Y, Glaser SS, Marzioni M, Phinizy JL, Francis H, Lesage G. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology 34: 868–876, 2001. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources