PROLIFERATION OF HEPATIC STELLATE CELLS IS INHIBITED BY PHOSPHORYLATION OF CREB ON SERINE-133

Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation o f activated primary rat stellate cells was blocked by elevation of cAM P with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhib itor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-2318 7. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) a ctivation, we tested whether CREB-PSer133 was essential for stellate c ell quiescence, Nuclear extracts from quiescent, but not from activate d, stellate cells contained CREB-PSer133. Moreover, the phosphorylatio n of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and eith er a constitutively active PKA or a constitutively active CAMK-II inhi bited the proliferation of activated stellate cells, In contrast, expr ession of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, su ggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle, Conversely, expression of a trans-dominant negative CREB-Ala 1 33 mutant (which competes with CREB/CREB-PSer133 for cognate DNA bindi ng sites and presumably for protein interactions) induced a greater th an fivefold entry into S-phase of quiescent stellate cells, compared w ith control cells expressing either beta-galactosidase or wt CREB, ind icating that CREB-PSer133 may be indispensable for the quiescent stell ate cell phenotype, This study suggests that PKA and CAMK-II play an e ssential role on stellate cell activation through the induction of CRE B phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in patients with chronic liver dise ases.