Mullerian inhibiting substance regulates NFkappaB signaling and growth of mammary epithelial cells in vivo.

Müllerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro through interference with cell cycle progression and induction of apoptosis, a process associated with NFkappaB activation and up-regulation of one of its important target genes, IEX-1S (Segev, D. L., Ha, T., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., MacLaughlin, D. T., Donahoe, P. K., and Maheswaran, S. (2000) J. Biol. Chem. 275, 28371-28379). Here we demonstrate that MIS activates the NFkappaB signaling cascade, induces IEX-1S mRNA, and inhibits the growth of MCF10A, an immortalized human breast epithelial cell line with characteristics of normal cells. In vivo, an inverse correlation was found to exist between various stages of mammary growth and MIS type II receptor expression. Receptor mRNA significantly diminished during puberty, when the ductal system branches and invades the adipose stroma and during the expansive growth at lactation, but it was up-regulated during involution, a time of regression and apoptosis. Peripartum variations in MIS type II receptor expression correlated with NFkappaB activation and IEX-1S mRNA expression. Administration of MIS to female mice induced NFkappaB DNA binding and IEX-1S mRNA expression in the breast. Furthermore, exposure to MIS in vivo increased apoptosis in the mouse mammary ductal epithelium. Thus, MIS may function as an endogenous hormonal regulator of NFkappaB signaling and growth in the breast.

Mechanisms of mitogen-activated protein kinase activation in experimental diabetes.

Various growth factors and vasoactive substances are implicated in the pathogenesis of renal growth seen in early diabetes mellitus (DM). Mitogen-activated protein kinase (MAPK) is an important mediator of these extracellular stimuli. Protein kinase C (PKC), an enzyme known to be stimulated in DM, also activates MAPK. Thus, MAPK activity was examined in glomeruli from streptozotocin-induced DM rats. MAPK activity, measured as myelin basic protein kinase, was elevated by approximately 50% in DM versus controls (CON). Increased protein contents of p42mapk and p44mapk, as well as increased tyrosine phosphorylation and mobility shift of p42mapk, were also observed in DM. Tyrosine dephosphorylation of pp42mapk, on the other hand, assessed by incubating glomerular membrane with or without sodium orthovanadate (vanadate), was significantly diminished in DM. Protein expression of MAPK phosphatase-1 (MKP-1), a dual specificity phosphatase that inactivates MAPK, was approximately 60% of CON. Reduction in MKP-1 was reproduced in cultured mesangial cells grown under high glucose (30 mM; HG). The suppression of MKP-1 was PKC-dependent since incubation of HG cells with phorbol 12-myristate 13-acetate for 24 h abolished it. Furthermore, calcium ionophore A23187 reversed the suppression, suggesting that blunted Ca2+ signalling, characteristic of HG cells secondary to PKC stimulation, may be the cause. These results demonstrate that glomerular MAPK is activated in DM by multiple mechanisms i.e., increases in protein contents, increased phosphorylation, and decreased dephosphorylation of the enzyme due to suppression of MKP-1. These alterations may have an implication in the pathogenesis of diabetic nephropathy.

Trapidil inhibits platelet-derived growth factor-stimulated mitogen-activated protein kinase cascade.

Trapidil, an antiplatelet drug, has been shown to reduce restenosis after angioplasty. It exerts its action, at least in part, by inhibiting vascular smooth muscle cell proliferation, antagonizing platelet-derived growth factor (PDGF). We examined its site of action on PDGF cellular signaling. Exposure of cultured rat vascular smooth muscle cells to increasing concentrations of trapidil for 18 hours resulted in a dose-dependent reduction in PDGF-BB-stimulated [3H] thymidine incorporation. Trapidil (400 microg/mL) increased PDGF beta-receptor protein by 28+/-8%, whereas PDGF-induced tyrosine phosphorylation of PDGF beta-receptor remained unchanged. PDGF-induced tyrosine phosphorylation of phospholipase Cgamma, the p85 regulatory subunit of phosphatidyl-inositol 3 kinase, Ras GTPase-activating protein, and an adaptor molecule Shc were also not altered. On the other hand, trapidil inhibited PDGF-stimulated mitogen-activated protein kinase (MAP kinase) activity by 35+/-7% at 10 minutes and by 32+/-10% at 6 hours. Activation of Raf-1, an upstream activator of MAP kinase, by PDGF was also attenuated by trapidil. Moreover, protein content of MAP kinase phosphatase-1, which inactivates MAP kinase, was elevated in trapidil-treated cells. These actions of trapidil may be mediated by cAMP. Thus, there was a 1.9-fold increase in cellular cAMP generation in trapidil-treated cells. The present results demonstrate that trapidil antagonizes PDGF-induced mitogenesis and MAP kinase activation in vascular smooth muscle cells, probably through cAMP.