Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes

Abstract

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.

Structure, Regulation and Function of Gap Junctions in Liver

“…Similar alterations are seen in Cx32 expression, whereas Cx43 production remains unchanged (Kren et al, 1993; Temme et al, 2000; Traub et al, 1983). These findings can be reproduced in an in vitro model of hepatocyte proliferation, namely mitogen-stimulated primary hepatocytes (Fladmark et al, 1997; Kojima et al, 1997; Kojima et al, 2004). In this system, mitogen-activated protein kinase phosphorylates Cx32, resulting in its decreased expression (Kojima et al, 2004).…”

“…These findings can be reproduced in an in vitro model of hepatocyte proliferation, namely mitogen-stimulated primary hepatocytes (Fladmark et al, 1997; Kojima et al, 1997; Kojima et al, 2004). In this system, mitogen-activated protein kinase phosphorylates Cx32, resulting in its decreased expression (Kojima et al, 2004). In fact, connexin phosphorylation seems to be a major mechanism underlying GJIC alterations in liver cell cycling.…”

p38 MAP-kinase regulates function of gap and tight junctions during regeneration of rat hepatocytes

“…In a recent study, protein expression of tight junction components such as claudin-3, ZO-1 and ASIP was found to increase after PH [21]. We previously found that the PI3-kinase pathway rather than the MAPkinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of gap junction protein Cx32, but not tight junction protein claudin-1 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP-kinase [22]. However, the detail mechanisms, including other signal transduction pathways, for regulation of gap and tight junctions during liver regeneration are as yet unclear.…”

Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes

“…In particular, it is essential for the proper differentiation of stem cells in hematopoietic [ 23 ], muscular [ 24 ], neural [ 25 , 26 ], cardiac [ 27 ], pancreatic [ 28 – 30 ], lung [ 31 ], and skin [ 32 , 33 ] tissues. Former studies showed that inhibition of p38 MAPK activity could regulate Cx32 or Cx43 in both rat neuronal stem cells and liver epithelial cells [ 34 – 36 ]. Moreover, in vivo research indicated that downregulation of Cx32 protein after partial hepatectomy could be reversed by the treatment of p38 MAPK inhibitor [ 37 ].…”