Abstract
Small cholangiocytes proliferate via activation of calcium (Ca(2+) )-dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate-dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca(2+) -dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an α(1) -adrenergic agonist stimulating intracellular D-myo-inositol-1,4,5-triphosphate (IP(3) )/Ca(2+) levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP(3) /Ca(2+) -dependent signaling. Phenylephrine stimulated the production of intracellular IP(3) . The Ca(2+) -dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca(2+) -dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca(2+) -dependent activation of NFAT2 and Sp1. CONCLUSION: Selective stimulation of Ca(2+) -dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins.