Overexpression and amplification of hepatocyte growth factor (HGF) receptor (Met) have been detected in many types of human cancers, suggesting a critical role for Met in growth and development of malignant cells. However, the molecular mechanism by which Met contributes to tumorigenesis is not well known. The tyrosine kinase c-Src has been implicated as a modulator of cell proliferation, spreading, and migration; these functions are also regulated by Met. To explore whether c-Src kinase is involved in HGF-induced cell growth, a mouse mammary carcinoma cell line (SP1) that co-expresses HGF and Met and a nonmalignant epithelial cell line (Mv1Lu) that expresses Met but not HGF were used. In this study, we have shown that c-Src kinase activity is constitutively elevated in SP1 cells and is induced in response to HGF in Mv1Lu cells. In addition, c-Src kinase associates with Met following stimulation with HGF. The enhanced activity of c-Src kinase also correlates with its ability to associate with Met. Expression of a dominant negative double mutant of c-Src (SRC-RF), lacking both kinase activity (K295R) and a regulatory tyrosine residue (Y527F), in SP1 cells significantly reduced c-Src kinase activity and strongly blocked HGFinduced motility and colony growth in soft agar. In contrast, expression of the dominant negative c-Src mutant had no effect on HGF-induced cell proliferation on plastic. Taken together, our data strongly suggest that HGFinduced association of c-Src with Met and c-Src activation play a critical role in HGF-induced cell motility and anchorage-independent growth of mammary carcinomas and further support the notion that the presence of paracrine and autocrine HGF loops contributes significantly to the transformed phenotype of carcinoma cells.

Stromal cells can dramatically affect the growth and metastatic capability of breast carcinoma cells. Growth factors, considered to be important mediators of this process, act as either mitogenic or mito-inhibitory regulators. We have developed an in vitro coculture system to examine the influence of adipocytes, a dominant mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have reported that conditioned medium (CM) from 3T3-L1 adipocytes can promote in vitro growth of SP1 cells. We now show that the major mitogenic signal derived from 3T3-L1 adipocyte CM is mediated by hepatocyte growth factor (HGF). Neutralizing antibody against HGF at 15 micrograms/ml completely abrogated mitogenic activity of 3T3-L1 CM. Furthermore, heparin, an inhibitor of biological activity of HGF, inhibited the mitogenic activity of 3T3-L1 CM. Western blot analysis also confirmed the presence of HGF in 3T3-L1 CM. Although basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) were mitogenic for SP1 cells, neutralizing antibodies against IGF-I, bFGF, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) did not inhibit the mitogenic activity of 3T3-L1 CM. Immunoprecipitation and immunoblotting of HGF receptor/c-met showed that c-met is expressed at high level in SP1 cells, and is phosphorylated following HGF ligation. Together, our present data demonstrate that 3T3-L1 adipocytes secrete HGF, which stimulates SP1 cell growth by a paracrine mechanism. Furthermore, the mitogenic effect of 3T3-L1 CM requires HGF receptor ligation and activation of tyrosine kinase signaling cascades in SP1 cells. These results highlight the importance of stromal-tumor cell interactions and suggest that HGF secreted by adipocytes may be a key regulator of mammary tumor growth.

Recent studies have demonstrated the importance of beta1 integrin in oligodendrocyte maturation in vitro. Similar studies in vivo have been difficult due to the embryonic and perinatal lethality of null mutations in integrin subunits. Here, we have generated transgenic mouse models that overexpress full length beta1 integrin or express a dominant-negative beta1 integrin DeltaC (lacking the C-terminal tail) under the control of the proteolipid protein (PLP) promoter. We demonstrate that these transgenes are expressed predominantly in CNS tissues and more specifically in oligodendrocytes. Further analysis reveals that the dominant-negative beta1 integrin DeltaC transgenic mice, but not the full length beta1 integrin mice, have hypomyelinated axons in spinal cords and optic nerves. In addition, there is a significant increase in the number of unmyelinated axons within the spinal cords and optic nerves of the beta1 integrin DeltaC mice. In contrast, the corpus callosum from these mice did not show similar myelin defects. To assess if remyelination would be affected in the corpus callosum, mice were subjected to a cuprizone-induced demyelination. Interestingly, the dominant-negative mice recovered from this insult in a manner similar to the wild type littermates. Axons within the corpus callosum that were remyelinated had normal g-ratios; however, the actual percentage of myelinated axons was significantly reduced compared with wild type mice. We also show that the defects observed in the dominant-negative beta1 integrin DeltaC mice are accompanied by disruption of the MAP-kinase signaling pathway. Our work highlights the importance of beta1 integrin-mediated signaling in CNS myelination in vivo.

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Dystonia musculorum (dt) is an autosomal recessive sensory neuropathy in mice resulting from a mutation in the gene encoding the cytoskeletal linker protein Bpag1. In addition to neurodegeneration, dt mice display myelination abnormalities in the peripheral nervous system. In this report we investigated whether myelination abnormalities are also present in the central nervous system of dt(Tg4) mice. Transcripts for both neural isoforms of Bpag1 (a1 and a2) were detected in optic nerves and spinal cords of wild-type mice. Light microscopy of resin-embedded thin sections revealed a reduction in myelinated axons in both optic nerves and spinal cords in dt(Tg4) mice. As well, hypermyelinated axons were detected in these tissues. Ultrastructural analysis of optic nerves and spinal cords from dt(Tg4) mice revealed an increase in the number of amyelinated axons, the presence of hypo- and hypermyelinated axons, and redundant myelin that course away from axons. Changes in the level of myelin proteins accompanied the morphological alterations. Myelin-associated glycoprotein levels were reduced in optic nerves of dt(Tg4) mice, and myelin basic protein levels were altered in optic nerves, sciatic nerves, and spinal cords of affected mice. Short-term cultures of oligodendrocytes derived from dt(Tg4) mice did not show morphological alterations.

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