SMAD3 Knockout Cell Lysate

Serum prolactin levels gradually increase from birth to puberty in both male and female rats, with higher levels observed in female since the first days of life. The increase in lactotroph secretion was attributed to the maturation of prolactin-inhibiting and prolactin-releasing factors; however, those mechanisms could not fully explain the gender differences observed. Prolactin secretion from isolated lactotrophs, in the absence of hypothalamic control, also increases during the first weeks of life, suggesting the involvement of intra-pituitary factors. We postulate that pituitary transforming growth factor beta 1 (TGF beta 1) is involved in the regulation of prolactin secretion as well as in the gender differences observed at early postnatal age. Several components of the local TGF beta 1 system were evaluated during postnatal development (11, 23, and 45 days) in female and male Sprague-Dawley rats. In vivo assays were performed to study local TGF beta 1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGF beta 1, concomitant with the highest expression of TGF beta 1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGF beta 1 levels only in females. Dopamine and estradiol induce TGF beta 1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGF beta 1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGF beta 1 on prolactin secretion is gender specific; and (3) this mechanism is responsible, at least partially, for the gender differences observed being relevant during postnatal development.

Large-conductance and Ca2+-activated K+ (BK) channels are expressed in human hepatic stellate cells (HSCs), where they have roles in normal hepatic microcirculation, as well as in portal hypertension in liver cirrhosis through the regulation of contractility in activated HSCs. Nevertheless, whether BK channel activity exerts protective effects against aberrant HSC activation and hepatic fibrosis is unknown. Here, we report that BK channels are expressed in activated primary rat HSCs as well as in a human HSC line. Moreover, whole-cell K+ currents recorded from activated HSCs were markedly increased by exposure to rottlerin, a BK channel-specific activator, but were inhibited by treatment with the BK channel-specific inhibitor, paxilline, suggesting that BK channels are functional in activated HSCs. Overexpression but not downregulation of the BK channel pore-forming alpha subunit, KCNMA1, led to reduced migration and collagen expression in activated HSCs. Consistently, rottlerin treatment suppressed the fibrogenic cell function both in vitro and in CCl4-induced liver fibrosis in vivo. Microarray and pathway analysis, combined with a luciferase reporter assay and western blotting, further showed that rottlerin treatment led to a significant downregulation of the profibrotic TGF beta 1/SMAD3 and JAK/STAT3 signaling pathways, both in vitro and in vivo. Our findings not only link BK channel function to profibrotic signaling pathways, but also provide evidence that BK channel activation represents a promising therapeutic strategy for the treatment of liver fibrosis.