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The most prominent cell surface attachment site for intermediate filaments (IF) at epithelial cell-cell interfaces is the desmosome (reviewed in Collins and Garrod, 1994; Schmidt et al., 1994; Garrod et [...]

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin [E.sub.2] ([PGE.sub.2]) and a 3.4- to 6.5-fold increase in prostacyclin ([PGI.sub.2]) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of [PGE.sub.2] 1.7- to 1.9-fold and [PGI.sub.2] 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of [PGE.sub.2] and [PGI.sub.2] from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more [PGE.sub.2] and 3.8-fold more [PGI.sub.2] released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more [PGE.sub.2] and 11.2-fold more [PGI.sub.2] released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC. cyclooxygenase; microfilaments; microtubules; vasoprotection; prostaglandin [E.sub.2]; prostacyclin

Studies the regulation of prostaglandin and prostacyclin release from human umbilical vein endothelial cells by actin cytoskeleton. Cell viability and apoptosis detection; Effect of cytoskeletal disruption on prostaglandin synthesis and release; Influence of cytosksleton disruption on cyclooxygenase-2 protein levels.

Maintenance of optimal bone physiology requires the coordinated activity of osteoclasts that resorb old bone and osteoblasts that deposit new bone. Mechanical loading of bone and the resulting movement of interstitial fluid within the spaces surrounding bone cells is thought to play a key role is maintaining optimal bone mass. One way in which fluid movement may promote bone formation is by enhancing osteoblast survival. We have shown previously that application of fluid flow to osteoblasts in vitro confers a protective effect by inhibiting osteoblast apoptosis (Pavalko et al., 2003, J. Cell Physiol., 194: 194-205). To investigate the cellular mechanisms that regulate the response of osteoblasts to fluid shear stress, we have examined the possible interaction between fluid flow and growth factors in MC3T3-E1 osteoblast-like cells. We found that insulin-like growth factor-I (IGF-I) was significantly more effective at preventing TNF-alpha-induced apoptosis when cells were first subjected to mechanical loading by exposure to either unidirectional or oscillatory fluid flow compared to cells that were maintained in static culture. Additionally, downstream signaling in response to treatment with IGF-I, including ERK and Akt activation, was enhanced in cells that were subjected to fluid flow, compared to cells maintained in static culture. Furthermore, we found that PKC activity is essential for fluid shear stress sensitization of IGF-IR, since a specific inhibitor of PCKzeta function blocked the flow-enhanced IGF-I-activated Akt and ERK phosphorylation. Together, our results suggest that fluid shear stress may regulate IGF-I signaling in osteoblasts in a PKC-zeta-dependent manner.