Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca2+-dependent PKC isoforms - PubMed (original) (raw)
Comparative Study
doi: 10.1186/1472-6793-7-6.
Shannon Glaser, Heather Francis, Sharon DeMorrow, Yoshiyuki Ueno, Domenico Alvaro, Luca Marucci, Marco Marzioni, Giammarco Fava, Julie Venter, Shelley Vaculin, Bradley Vaculin, Ian Pak-Yan Lam, Vien Hoi-Yi Lee, Eugenio Gaudio, Guido Carpino, Antonio Benedetti, Gianfranco Alpini
Affiliations
- PMID: 17640386
- PMCID: PMC1939715
- DOI: 10.1186/1472-6793-7-6
Comparative Study
Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca2+-dependent PKC isoforms
Silvia Taffetani et al. BMC Physiol. 2007.
Abstract
Background: Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca2+-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes.
Results: Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca2+]i levels and PKCbeta-I phosphorylation but decreased PKCalpha phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha.
Conclusion: We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.
Figures
Figure 1
The localization of prolactin receptor in the liver was evaluated by immunohistochemistry (scale bar = 50 μm) in liver sections from normal and BDL female and male rats. Bile ducts from normal and BDL female and male rats express these receptors (arrows). No immunohistochemical reaction was observed when a consecutive liver section of the same field was incubated with non-immune serum. Hepatocytes from normal and BDL female and male rats express the prolactin receptor.
Figure 2
The localization of prolactin receptor in the liver was evaluated by immunofluorescence (scale bar = 20 μm) in liver sections from normal and BDL female and male rats. By immunofluorescence, prolactin receptor immunoreactivity (red) was co-localized with CK-19 immunoreactivity (green; indicated by arrows) demonstrating cholangiocyte expression; sections were counterstained with DAPI; in the merged photograph we show co-localization of prolactin receptor and CK-19. Hepatocytes from normal and BDL female and male rats express the prolactin receptor.
Figure 3
Real time PCR for the message for the short and long form of prolactin receptor in total cholangiocyte RNA (0.75 μg) from normal and BDL female rats. Normal female cholangiocytes express both the short and long form of prolactin receptor mRNA (expressed as ratio to GAPDH mRNA); following BDL, the expression of both the short and long form of prolactin receptor mRNA (expressed as ratio to GAPDH mRNA) significantly increased in purified cholangiocytes. Data are mean ± SEM of 3 experiments. *p < 0.05 vs. relative expression of short and long prolactin receptor mRNA of normal cholangiocytes. NR = normal rat; PRLR = prolactin receptor.
Figure 4
Measurement of the number of [top panel] PCNA- and [lower panel] CK-19-positive cholangiocytes in liver sections (5 μm, 3 slides analyzed per group) and [c] PCNA protein expression in purified female cholangiocytes from NaCl- or prolactin-treated rats. Administration of prolactin to normal female rats increased the number of PCNA-positive cholangiocytes (arrows) and CK-19-positive cholangiocytes compared with normal rats treated with NaCl. Orig. magn., ×20 (PCNA) and ×10 (CK-19). Data are mean ± SEM of 5 values obtained from the 3 slides evaluated per each group of animal. * p < 0.05 vs. the corresponding value of NaCl-treated rats.
Figure 5
Determination of [Ca2+]i levels in female normal rat cholangiocytes treated with 0.2% BSA or 100 nM prolactin. Prolactin induced a sustained increase in [Ca2+]i levels compared with cholangiocytes treated with 0.2% BSA [top panel]. Data are mean ± SEM of 4 experiments. * p < 0.05 vs. the corresponding basal values. [lower panel] A calcium tracing, which is the average of three independent measurements, is shown in shown. As the tracing shows there is no change in fluorescence during the basal measurement period that demonstrates that the cells do not leak as influx of extracellular calcium would alter fluorescence. Cholangiocyte responsiveness to the Ca2+ ionophore, ionomycin, is shown.
Figure 6
In vitro effect of prolactin on the phosphorylation of Ca2+-dependent PKC isoforms. Immunoblots for PKC-α, PKC-β-I, PKC-β-II and PKC-γ in normal female cholangiocytes stimulated for 90 minutes at 37°C with 0.2% BSA (basal value) or prolactin (100 nM) with 0.2% BSA. When cholangiocytes were treated with prolactin, there was an increase in the phosphorylation of PKCβ-I and a marked decrease in PKCα phosphorylation; no significant changes in the phosphorylation of PKCβ-II and PKCγ were observed in normal female cholangiocytes treated with prolactin or 0.2% BSA. Data are mean ± SEM of 3 experiments. * p < 0.05 vs. corresponding basal values. PKC = protein kinase C.
Figure 7
Administration of anti-prolactin antibody to female BDL rats decreased the number of [top panel] PCNA-positive cholangiocytes and [lower panel] CK-19-positive cholangiocytes compared to cholangiocytes from BDL female rats treated with non-immune serum. Orig. magn., ×20 (PCNA) and ×10 (CK-19). Data are mean ± SEM of 5 values obtained from the 3 slides evaluated per each group of animal. * p < 0.05 vs. the corresponding value of BDL rats treated with non-immune serum.
Figure 8
Immunohistochemistry for prolactin in liver sections of normal and BDL female rats shows that intrahepatic bile ducts express the protein for prolactin (arrows). Bar = 50 μm.
Figure 9
Real time PCR for prolactin mRNA in total RNA from normal and BDL female cholangiocytes. We demonstrated that: (i) female normal cholangiocytes express prolactin mRNA at low levels; and (ii) following BDL, prolactin mRNA markedly increased in female cholangiocytes. Data are mean ± SEM of 3 experiments. *p < 0.05 vs. relative expression of prolactin receptor of normal female cholangiocytes.
References
- 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. 1998;274:G767–G775. - PubMed
- Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary epithelia. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby W, Schachter D, Shafritz DA, editor. The Liver; Biology & Pathobiology. 4. Philadelphia, PA: Lippincott Williams & Wilkins; 2001. pp. 421–435.
- LeSage G, Glaser S, Ueno Y, Alvaro D, Baiocchi L, Kanno N, Phinizy JL, Francis H, Alpini G. Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis. Am J Physiol Gastrointest Liver Physiol. 2001;281:G182–G190. - PubMed
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