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The Rho family GTPases are key signalling components that regulate the cytoskeleton and adhesion during cell migration. The Vav family of proteins act as guanine nucleotide exchange factors for several Rho GTPases. There are three isoforms of Vav expressed in mammalian cells: Vav1, the expression of which is largely restricted to haematopoietic cells, Vav2 and Vav3. In this study the role of Vav proteins in macrophage migration has been investigated using macrophages derived from mice lacking one or all three Vav isoforms. Cell migration and morphology were not significantly affected when single isoforms of Vav were absent from macrophages. However macrophages lacking all three isoforms adopted an elongated morphology in culture, which resulted in more persistent cell migration. Vav proteins were not required for chemotaxis to the macrophage chemo-attractant, colony-stimulating factor-1 (CSF-1). Vav proteins were also not required for CSF-1-stimulated Rac1 activation or Rac-mediated cytoskeletal reorganization in response to CSF-1. However, in response to CSF-1 stimulation Vav1 and Vav3 were phosphorylated on tyrosine residues, which has previously been shown to regulate their GEF catalytic activity. Macrophages lacking all three Vav proteins were defective in spreading upon adhesion to both glass and plastic. The defect correlated with reduced activation of Rac1 and RhoA, and a reduction in the activation of Erk1/2 and phosphorylation of paxillin in response to adhesion. Vav proteins are therefore not required for directed macrophage migration to the chemo-attractant CSF-1, but have an important role in adhesion-dependent signalling and are needed to maintain normal macrophage migration and morphology.

Id2 is a HLH factor with well established role in cellular proliferation and differentiation. Traditionally Id2 is classified as a dominant negative member of the HLH family, yet it shows broad association with proteins from other families. Changes in its expression in response to hormonal and growth signal differs greatly between cell types though little is known about its regulation in macrophages. In this study the regulation of Id2 protein expression by glucose and hormones is studied in J774.2 macrophages. In J774.2 cells glucose induces increases in protein levels of Id2. Up regulation of Id2 requires glutamine, is mimicked by glucosamine and is inhibited by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT). Further, adenoviral mediated overexpression of GFAT increases levels of Id2. We conclude that hexosamine pathway mediate changes in Id2 level. Effect of glucose is cell type specific. While a similar effect is observed in Hepatocyte, little or no changes occur in L6 Myocytes and 3T3-L1 Adipocytes. Id2 acts as negative regulator of transcription by forming inactive heterodimer with other members of the HLH family such as SREBP-1. Previously, work in the laboratory has shown that high levels of glucose prime J774.2 macrophages in such a way that insulin and leptin are able to reduce expression of hormone sensitive lipase (HSL). Here we observe that activity of mouse HSL promotor is increased when co-expressed with SREBP-1. The SREBP-1 induced increase in HSL promoter activity is attenuated upon co-expression with Id2, indicating that increased Id2 levels can mimic the effects of high glucose. Id2 expression in J774.2 cells is affected by intracellular cAMP raising agents and insulin. Effect of cAMP is mediated by Epac through PI3 kinase dependent pathway. GSK3 is the end effector of both cAMP and insulin effect, its inactivation leads to up regulation of Id2 protein level.

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