We recently identified 1K K,25-dihydroxy-3-epi-vitamin D Q as a major in vitro metabolite of 1K K,25-dihydroxyvitamin D Q , produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1K K,25-dihydroxy-3-epi-vitamin D Q from the serum of rats treated with pharmacological doses of 1K K,25-dihydroxyvitamin D Q . 1K K,25-dihydroxy-3-epi-vitamin D Q was identified through its co-migration with synthetic 1K K,25-dihydroxy-3-epi-vitamin D Q on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1K K,25-dihydroxy-3-epi-vitamin D Q , other previously known metabolites, namely, 1K K,24(R),25-trihydroxyvitamin D Q , 1K K,25-dihydroxy-24-oxo-vitamin D Q and 1K K,25-dihydroxyvitamin D Q -26,23-lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1K K,25-dihydroxy-3-epi-vitamin D Q is an in vivo metabolite of 1K K,25-dihydroxyvitamin D Q in rats.z 1999 Federation of European Biochemical Societies.

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IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.