TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Mediation of aldose reductase in lipopolysaccharide-induced inflammatory signals in mouse peritoneal macrophages

Aldose reductase (AR; AKR1B1) a member of aldo-keto reductase super family, that we had shown earlier mediates cytotoxic signals induced by high glucose, cytokines and growth factors, also mediates the inflammatory signals induced by Gram-negative bacterial endotoxin, lipopolysaccharide (LPS). Inhibition of AR by three distinct AR inhibitors sorbinil, tolrestat or zopolrestat suppressed the LPS-induced production of inflammatory cytokines such as TNF-alpha, IL-6, IL-1beta, IFN-gamma, and chemokine MCP-1 in murine peritoneal macrophages. Inhibition of AR also prevented the production of nitric oxide, and prostaglandin E2 and expression of iNOS and Cox-2 proteins. The LPS-induced DNA binding activity of NF-kappaB and AP1 were significantly inhibited by AR inhibitors, and this effect was mediated through the inhibition of phosphorylation of IkappaB-alpha, IKK alpha/beta and PKC. These results suggest the therapeutic use of AR inhibitors as anti-inflammatory drugs.     

几种扩血管多肽对bFGF促血管平滑肌细胞增殖作用的影响

目的和方法：研究肾上腺髓质素（Ａｄｍ）、降钙素基因相关肽（ＣＧＲＰ）及Ｃ－型心房利太（ＣＮＰ）对碱性成纤维细胞生长因子（ｂＦＧＦ）促血管平滑细胞（ＶＳＭＣ）增殖作用的影响及其机制。结果：孵育２４ｈ后,ｂＦＧＦ刺激ＶＳＭＣ增殖较对照组增加２．１倍（Ｐ〈０．０１）,细胞内蛋白磷酸化程度增加１．４倍（Ｐ〈０．０１）,ＰＫＣ及ＭＡＰＫ活性分别增加１．５和２．５倍（Ｐ〈０．０１０;Ａｄｍ．ＣＧＲＰｔ ＣＮＰ     

Protein kinase C alpha and zeta regulate nitric oxide-induced NF-kappa B activation that mediates cyclooxygenase-2 expression and apoptosis but not dedifferentiation in articular chondrocytes

Nitric oxide (NO) regulates differentiation, survival, and cyclooxygenase (COX)-2 expression in articular chondrocytes. NO-induced apoptosis and dedifferentiation are mediated by p38 kinase activity and p38 kinase-independent and -dependent inhibition of protein kinase C (PKC)alpha and zeta. Because p38 kinase also activates NF-kappa B, we investigated the functional relationship between PKC and NF-kappa B signaling and the role of NF-kappa B in apoptosis, dedifferentiation, and COX-2 expression. We found that NO-stimulated NF-kappa B activation was inhibited by ectopic PKC alpha and zeta expression, whereas NO-stimulated inhibition of PKC alpha and zeta activity was not affected by NF-kappa B inhibition. Inhibition of NO-induced NF-kappa B activity did not affect inhibition of type II collagen expression but did abrogate COX-2 expression and apoptosis. Taken together, our results indicate that NO-induced inhibition of PKC alpha and zeta activity is required for the NF-kappa B activity that regulates apoptosis and COX-2 expression but not dedifferentiation in articular chondrocytes.     

A dominant negative protein kinase C zeta subspecies blocks NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a number of genes. NF-kappa B is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein I kappa B. Following stimulation of cells, I kappa B dissociates from NF-kappa B, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-kappa B activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-kappa B. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system of Xenopus laevis oocytes that the PMA-insensitive PKC isotype, zeta PKC, is a required step in the activation of NF-kappa B in response to ras p21. We demonstrate here that overexpression of zeta PKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-kappa B into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of zeta PKC dramatically inhibits the kappa B-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that zeta PKC plays a decisive role in NF-kappa B regulation in mammalian cells.     

Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta.

Protein kinase C zeta (zeta PKC) is critically involved in the control of a number of cell functions, including proliferation and nuclear factor kappa B (NF-kappa B) activation. Previous studies indicate that zeta PKC is an important step downstream of Ras in the mitogenic cascade. The stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase (MAPK), which is required for cell growth. MAPK is activated by phosphorylation by another kinase named MAPK kinase (MEK), which is the substrate of a number of Ras-activated serine/threonine kinases such as c-Raf-1 and B-Raf. We show here that MAPK and MEK are activated in vivo by an active mutant of zeta PKC, and that a kinase-defective dominant negative mutant of zeta PKC dramatically impairs the activation of both MEK and MAPK by serum and tumour necrosis factor (TNF alpha). The stimulation of other kinases, such as stress-activated protein kinase (SAPK) or p70S6K, is shown here to be independent of zeta PKC. The importance of MEK/MAPK in the signalling mechanisms activated by zeta PKC was addressed by using the activation of a kappa B-dependent promoter as a biological read-out of zeta PKC.     

Activation of NF-kappa B in vivo is regulated by multiple phosphorylations.

The activation of nuclear factor kappa B (NF-kappa B) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-kappa B/I kappa B components following stimulation and show that the final step of NF-kappa B induction in vivo involves phosphorylation of several members of the NF-kappa B/I kappa B protein families. In HeLa cells as well as in B cells, TNF-alpha rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-kappa B precursors and I kappa B alpha became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-kappa B revealed constitutively phosphorylated p65 and I kappa B alpha. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of I kappa B alpha. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-kappa B, as has been shown before for I kappa B alpha. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-alpha selectively activates only specific NF-kappa B heteromers and that modifications regulate not only I kappa B molecules but also NF-kappa B molecules. In fact, cellular NF-kappa B activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-kappa B was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-kappa B translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-alpha. Thus, phosphorylation appears to be a general mechanism for activation of NF-kappa B in vivo.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.