Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P 3 ), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P 3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-␣-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C 16 -PtdIns-3,4,5-P 3 ) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C 16 -PtdIns-3,4,5-P 3 . These results are consistent with previous observations that PtdIns-3,4,5-P 3 activates Ca 2؉ -independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P 3 appears to directly initiate cellular motility via activation of a PKC family member.Initiation of cellular motility has been demonstrated with multiple growth factors, including platelet-derived growth factor (PDGF) 1 (1), hepatocyte growth factor (HGF) (2), and insulin (3). The mechanisms whereby cells undergo chemotaxis (directional cell movement) and chemokinesis (random cell movement) are complex, requiring dissolution of cell-cell contacts (such as tight junctions in epithelial cells) and cell-surface contacts, formation of lamellipodia, actin filament severing and nucleation, and finally contraction of the actin filament network leading to movement of the cell body (4). An understanding of the signaling pathways required to orchestrate these cellular events should provide critical new insights into numerous biological events such as cell migration and organization during organ development and wound healing, tumor cell metastasis, and progression of arterial atherosclerotic plaques.Mutations in the PDGF receptor that eliminate binding of phosphoinositide 3-kinase PI 3-kinase-impair PDGF-dependent chemotaxis (1, 5, 6), and selective activation of the PI 3-kinase is sufficient to initiate motility (7). The lipid products of PI 3-kinase, PtdIns-3,4-P 2 , and PtdIns-3,4,5-P 3 are elevated acutely in response to PDGF (8) and are thought to act as second messengers (9 -11). Although the in vivo function of these lipids has not been demonstrated, they activate calciumindependent protein kinase C family members in a stereospecific manner (12-16). Thus, we investigated the possibility that PtdIns-3,4,5-P 3 stimulates cell motility via activation of a PKC family member. MATERIALS AND METHODSCell Culture and Reagents-The majority of experiments were performed with NIH 3T3 fibroblasts, using PDGF as the positive control. Selected experiments were repeated with mIMCD-3 cells, a murine renal tubular epithelial cell line that expresses the c-met receptor and exhibits striking chemotaxis to a gradient of HGF (17-19). All ...
The Bronx Aging Study is a 10-year prospective investigation of very elderly volunteers (mean age at study entry, 79 years; range, 75-85 years) designed to assess risk factors for dementia and coronary and cerebrovascular (stroke) diseases. Entry criteria included the absence of terminal illness and dementia. All subjects (n = 350) included in this report had at least two lipid and lipoprotein determinations. Overall, more than one third of subjects showed at least a 10% change in lipid and lipoprotein levels between the initial and final measurements. Moreover, mean levels for women were consistently different than those for men, and because of this finding subjects were classified into potential-risk categories based on the changes observed by using their sex-specific lipid and lipoprotein distributions. The incidences of cardiovascular disease, dementia, and death were compared between risk groups. Proportional-hazards analysis showed that in men a consistently low high density lipoprotein cholesterol level (less than or equal to 30 mg/dl) was independently associated with the development of myocardial infarction (p = 0.006), cardiovascular disease (p = 0.002), or death (p = 0.002). For women, however, a consistently elevated low density lipoprotein cholesterol level (greater than or equal to 171 mg/dl) was associated with myocardial infarction (p = 0.032). Thus, low high density lipoprotein cholesterol remains a powerful predictor of coronary heart disease risk for men even into old age, while elevated low density lipoprotein cholesterol continues to play a role in the development of myocardial infarction in women. The findings suggest that an unfavorable lipoprotein profile increases the risk of cardiovascular morbidity and mortality even at advanced ages for both men and women.
The association of hepatocyte growth factor (HGF) with its high-affinity receptor, c-met, has been shown to induce mitogenesis, motogenesis, and morphogenesis in renal epithelial cells (L. G. Cantley, E. J. G. Barros, M. Gandhi, M. Rauchman, and S. K. Nigam. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F271-F280, 1994), suggesting that HGF may be critical to the orchestration of both renal development and regeneration following injury. Although signal transduction pathways activated by c-met include the phosphatidylinositol 3-kinase (PI-3-kinase), phospholipase C gamma, ras, and others, the activation of PI-3-kinase has been the most striking in vivo. We therefore investigated whether the pathways that mediate phenotypic changes in inner medullary collecting duct cells are altered by inhibition of PI-3-kinase with the fungal metabolite, wortmannin. In these cells, the mean inhibitory concentration for in vitro wortmannin inhibition of PI-3-kinase was approximately 0.2 nM. At this low concentration, motogenesis (quantified by chemotaxis) and morphogenesis (by branching-process formation within collagen matrix) were inhibited in a striking and parallel fashion, while mitogenesis was inhibited to a lesser degree. These experiments suggest that activation of PI-3-kinase is critical for c-met-mediated chemotaxis and tubulogenesis.
Interaction of hepatocyte growth factor with its high affinity receptor c-met initiates a cascade of intracellular events leading to epithelial motility. An 11-amino acid sequence from the c-met receptor has been found to cause cell transformation in transfected fibroblasts (Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., Dalla, Z. P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. These findings demonstrate that the 11-amino acid sequence from c-met initiates epithelial motility via coincident activation of the PI 3-kinase and phospholipase C and that selective activation of the PI 3-kinase can initiate a partial chemotactic response.
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