Background Cardiovascular disease is the leading cause of death among diabetics. Vitamin D deficiency is associated with increased risk of cardiovascular disease in this population. To determine the mechanism by which vitamin D deficiency mediates accelerated cardiovascular disease in patients with diabetes, we investigated the effects of active vitamin D on macrophage cholesterol deposition. Methods and Results We obtained macrophages from 76 obese, diabetic, hypertensive patients with vitamin D deficiency (25-hydroxyvitamin D < 80 nmol/L)(group A) and four control groups: obese, diabetic, hypertensive patients with normal vitamin D (group B, n=15), obese, non-diabetic, hypertensive patients with vitamin D deficiency (group C, n=25), and non-obese, non-diabetic, non-hypertensive patients with vitamin D deficiency (group D, n=10) or sufficiency (group E, n=10). The same patient’s macrophages from all groups were cultured in vitamin D-deficient or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) supplemented media and exposed to modified low-density lipoprotein cholesterol. 1,25(OH)2D3 suppressed foam cell formation by reducing acetylated or oxidized low-density lipoprotein cholesterol uptake in diabetics only. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients accelerated foam-cell formation induced by modified LDL. 1,25(OH)2D3 downregulation of c-Jun N-terminal kinase activation reduced PPARγ expression, suppressed CD36 expression, and prevented oxLDL-derived cholesterol uptake. In addition, 1,25(OH)2D3 suppression of macrophage endoplasmic reticulum stress improved insulin signaling, downregulated SR-A1expression, and prevented oxLDL and AcLDL-derived cholesterol uptake. Conclusion These results identify reduced vitamin D receptor signaling as a potential mechanism underlying increased foam-cell formation and accelerated cardiovascular disease in diabetics.
The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP 2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP 2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP 2 rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can "wobble" when bound to the barbed end solely by the C-terminal "tentacle" of its -subunit.
Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.
Here we investigated the involvement of HS1, the hematopoietic cell-specific homolog of cortactin, in the actin-based functions of natural killer cells. Involvement of HS1 in T cell regulation has been established, as HS1 is required for the formation of immune synapses. 'Knockdown' of HS1 in natural killer cells resulted in defective lysis of target cells, cell adhesion, chemotaxis and actin assembly at the lytic synapse. Phosphorylation of the tyrosine residue at position 397 (Tyr397) was required for adhesion to the integrin ligand ICAM-1 and for cytolysis, whereas phosphorylation of Tyr378 was required for chemotaxis. Phosphorylation of Tyr397 was also required for integrin signaling and recruitment of integrins, adaptors and actin to the lytic synapse. Thus, HS1 is essential for signaling and actin assembly in natural killer cells, and the functions of the two phosphorylated tyrosine residues are distinct and separable.An emerging frontier in cell and systems biology is the relationship between signaling networks and the cytoskeleton. Signaling pathways control the assembly and activity of the cytoskeleton, and in many cases, cytoskeletal elements control signaling pathways through positive and negative feedback. Here we show that the cortactin homolog HS1 (also called HCLS1 or LckBP1; A001149), noted before as being important for the formation of immune synapses 1 , has a critical function as an integrator between signaling pathways and actin cytoskeletal regulation.The biology of natural killer (NK) cells in the innate immune system involves many receptormediated signaling and actin-assembly-based processes. Although much is known about these signaling and actin-assembly networks, relatively less is understood about how these two networks depend on and interact with each other. To address this issue, we studied HS1 as a candidate molecule for the transfer of information between the two networks.NK cells are large, granular lymphocytes that recognize and kill transformed and virus-infected cells. NK cells 'decide' the fate of potential target cells according to the balance of activating and inhibitory signals that result from receptor-ligand interactions between NK cells and target cells 2 . Most NK cells reside in the vasculature; thus, their cytolytic function begins with extravasation and chemotaxis toward target cells. These processes require integrin-mediated adhesion, signaling and actin assembly. When an NK cell encounters a potential target cell, NK receptors and integrins bind to ligands on the target cell surface; these interactions can lead to actin-mediated clustering of receptors, receptor-mediated signaling and the formation of a lytic synapse.Correspondence should be addressed to B.B. (boyd.butler@wustl.edu). Accession codes UCSD-Nature Signaling Gateway (http://www.signaling-gateway.org): A002871, A001209 and A001149. NIH Public Access Author ManuscriptNat Immunol. Author manuscript; available in PMC 2009 January 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Ma...
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