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Proinsulin C-peptide rapidly stimulates mitogen-activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3-kinase and pertussis toxin-sensitive G-protein.

ABSTRACT: It has been demonstrated that proinsulin C-peptide possesses several biological activities and that its specific binding sites are present on the surface of cell membranes. However, the molecular and cellular mechanisms of C-peptide actions are poorly known. In the present study we examined the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in C-peptide effects. C-peptide induced the phosphorylation of MAPK [p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2] in Swiss 3T3 and 3T3-F442A fibroblasts but not in 3T3-L1 fibroblasts and some other cell lines such as L(6)E(9) muscle cells. In Swiss 3T3 cells, C-peptide-induced phosphorylation of MAPK was dependent on time and concentration, being maximal at 1 min and at 1 nM C-peptide and was accompanied by an increase in MAPK activity and MAPK kinase (MEK) phosphorylation. The MAPK phosphorylation by C-peptide was abolished by treatment with pertussis toxin (PTX) and also with a MEK inhibitor, PD 98059. In addition, MAPK phosphorylation was attenuated by treatment with a phosphoinositide 3-kinase (PI-3K) inhibitor, wortmannin, and with a protein kinase C (PKC) inhibitor, GF109203X, and by down-regulation of PKC by prolonged treatment with PMA. Similar effects of the inhibitors and PTX were found on the MAPK phosphorylation induced by neuropeptide Y. These results suggest that C-peptide activates MAPK through a putative G(i)/G(o)-linked receptor for C-peptide and through PI-3K-dependent and PKC-dependent pathways.

SUBMITTER: Kitamura T

PROVIDER: S-EPMC1221719 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Project description:Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans.

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Proinsulin C-peptide rapidly stimulates mitogen-activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3-kinase and pertussis toxin-sensitive G-protein.

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