Progesterone Inhibits Endothelial Cell Migration Through Suppression of the Rho Activity Mediated by cSrc Activation.

We previously showed that progesterone (P4) could inhibit the proliferation of human umbilical venous endothelial cells (HUVECs) through the p53-dependent pathway. In the present study, we further demonstrated that P4 at physiologic levels (5-500 nM) concentration-dependently inhibited migration of HUVECs. This effect was blocked by pre-treatment with the P4 receptor (PR) agonist-antagonist, RU486, suggesting that the P4-induced migration inhibition in HUVECs was through the PR-mediated signaling pathway. Western blot analyses demonstrated that the levels of RhoA and Rac-1 protein were reduced in the P4-treated HUVECs. P4 also inhibited the membrane translocation of RhoA and Rac-1 protein. Moreover, the P4-induced migration inhibition in HUVECs was prevented by over-expression of the constitutively active RhoA construct (RhoA V14). However, pre-treatment with the ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the over-expression of RhoA-induced prevention effect on the P4-induced migration inhibition in HUVECs. These data suggest that the inhibition of Rho GTPases might account for the P4-induced migration inhibition of HUVECs. Pre-treatment with the cSrc inhibitor, PP2, prevented the P4-induced migration inhibition in HUVEC. The levels of phosphorylated focal adhesion kinase (FAK) and paxillin protein were also decreased by P4 treatment. Taken together, these results suggest that suppression of the Rho-mediated pathway might be involved in the signal transduction leading to the inhibition of cell migration caused by P4 in HUVECs.

L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF-κB translocation in cortical neurons/glia Cocultures.

In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS) to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C) affected neuroinflammation. LPS (100 ng/ml) induced the expression of inducible NO synthase (iNOS) and the production of NO, interleukin (IL)-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2) in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM) attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation) of mitogen-activated protein kinases (MAPKs), such as p38 at 30 min and extracellular signal-regulated kinases (ERKs) at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK) as these inhibitors. Vit. C also reduced LPS-induced IκB-α degradation and NF-κB translocation. Thus, Vit. C suppressed the LPS-stimulated production of inflammatory mediators in neuron/glia cocultures by inhibiting the MAPK and NF-κB signaling pathways.