Activation of alpha1-adrenergic receptors stimulate the growth of small mouse cholangiocytes Via calcium-dependent activation of nuclear factor of activated T cells 2 and specificity protein 1

Abstract

Small cholangiocytes proliferate via activation of Ca2+-dependent signaling in response to pathological conditions that trigger the damage of large cAMP-dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca2+-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 α1-adrenergic agonist stimulating intracellular IP3/Ca2+ 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 IP3/Ca2+-dependent signaling. Phenylephrine stimulated the production of intracellular IP3. The Ca2+-dependent transcription factors, NFAT2 and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca2+-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 shRNA. 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 Ca2+-dependent activation of NFAT2 and Sp1. Selective stimulation of Ca2+-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.

Epigenetic Regulation of Mir-34a Expression in Alcoholic Liver Injury

“…18,19 Briefly, cDNA was generated by reverse transcription using 1 g of total RNA isolated using the RNA isolation kit (Bio-Rad, Hercules, CA) and the SuperScript III Reverse Transcription Kit (Invitrogen). Quantitative real-time PCR was performed on a MX 3005P PCR Instrument (Stratagene, San Diego, CA) as described.…”

“…Quantitative real-time PCR was performed on a MX 3005P PCR Instrument (Stratagene, San Diego, CA) as described. 18,19 Each sample was tested in triplicate. The PCR products were verified by melting curve analysis and by 1.8% agarose gel electrophoresis of the PCR product.…”

Physiology of Cholangiocytes

“…Mz-Cha-1 cells, a human cholangiocarcinoma cell line, express four of seven P2X receptors (P2X 2 , P2X 3 , P2X 4 and P2X 5 ) (261). Normal rat cholangiocytes express P2X 2 , P2X 3 , P2X 4 and P2X 7 (70,71), with the P2X 4 receptor presumably being the dominant one (70).…”

The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells

“…Previous studies have shown that stimulation of a 1 -AR by the a 1 -AR agonist phenylephrine results in increased calcium transients, an effect that can be blocked by the use of the a 1 -AR antagonist prazosin (Thorlin et al 1998). In addition, glial a 1 -AR activation by NA promotes intracellular calcium release with downstream effects on membrane depolarisation, neurotransmitter release and cell proliferation (Legendre et al 1988;Stone et al 1999;Alpini et al 2011). It has also been demonstrated that a 1 -AR stimulation enhances glutamate uptake into cultured astrocytes (Hansson and Ronnback 1989;Fahrig 1993;Hertz et al 2010), and Ohashi et al (2007) showed that stimulation of a 1 -AR by phenylephrine suppressed stress-induced death and promoted cell survival in embryonic neural progenitor cells.…”