The human immunodeficiency virus type 1 Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated degradation of Ikappa B

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and induces its degradation by cytosolic proteasomes. This process involves the recruitment of human betaTrCP (TrCP), a key member of the SkpI-Cdc53-F-box E3 ubiquitin ligase complex that specifically interacts with phosphorylated Vpu molecules. Interestingly, Vpu itself, unlike other TrCP-interacting proteins, is not targeted for degradation by proteasomes. We now report that, by virtue of its affinity for TrCP and resistance to degradation, Vpu, but not a phosphorylation mutant unable to interact with TrCP, has a dominant negative effect on TrCP function. As a consequence, expression of Vpu in HIV-infected T cells or in HeLa cells inhibited TNF-alpha-induced degradation of IkappaB-alpha. Vpu did not inhibit TNF-alpha-mediated activation of the IkappaB kinase but instead interfered with the subsequent TrCP-dependent degradation of phosphorylated IkappaB-alpha. This resulted in a pronounced reduction of NF-kappaB activity. We also observed that in cells producing Vpu-defective virus, NF-kappaB activity was significantly increased even in the absence of cytokine stimulation. However, in the presence of Vpu, this HIV-mediated NF-kappaB activation was markedly reduced. These results suggest that Vpu modulates both virus- and cytokine-induced activation of NF-kappaB in HIV-1-infected cells.     

CGRP inhibits osteoprotegerin production in human osteoblast-like cells via cAMP/PKA-dependent pathway

The osteoprotegerin (OPG)/receptor activator of nuclear factor-B ligand (RANKL)/receptor activator of nuclear factor-B (RANK) system was evaluated as a potential target of CGRP anabolic activity on bone. Primary cultures of human osteoblast-like cells (hOB) express calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1, and, because CGRP stimulates cAMP (one of the modulators of OPG production in osteoblasts), it was investigated whether it affects OPG secretion and expression in hOB. CGRP treatment of hOB (10^–11 M–10^–7 M) dose-dependently inhibited OPG secretion with an EC₅₀ of 1.08 x 10^–10 M, and also decreased its expression. This action was blocked by the antagonist CGRP_8–37. Forskolin, a stimulator of cAMP production, and dibutyryl cAMP also reduced the production of OPG. CGRP (10^–8 M) enhanced protein kinase A (PKA) activity in hOB, and hOB exposure to the PKA inhibitor, H89 (2 x 10^–6 M), abolished the inhibitory effect of CGRP on OPG secretion. Conditioned media from CGRP-treated hOB increased the number of multinucleated tartrate-resistant acid phosphatase-positive cells and the secretion of cathepsin K in human peripheral blood mononuclear cells compared with the conditioned media of untreated hOB. These results show that the cAMP/PKA pathway is involved in the CGRP inhibition of OPG mRNA and protein secretion in hOB and that this effect favors osteoclastogenesis. CGRP could thus modulate the balance between osteoblast and osteoclast activity, participating in the fine tuning of all of the bone remodeling phases necessary for the subsequent anabolic effect. receptor-activity-modifying proteins; protein kinase A; osteoclast; cathepsin K     

Possible involvement of protein kinase C in parathyroid hormone degradation by osteoblast-like rat osteosarcoma cell line UMR106

The effects of 12-O-tetraadecanoyl phorbol-13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the parathyroid hormone (PTH) degrading activity in a PTH-responsive osteoblast-like rat osteosarcoma cell line UMR106 were investigated to assess the role of Ca2+-activated. Phospholipid dependent protein kinase (protein kinase C) on the degradation of hormones. TPA and OAG, activators of protein kinase C, enhanced the PTH degrading activity dose-dependently, whereas H-7, an inhibitor of protein kinase C, exhibited a dose-dependent inhibition on this activity. These data suggest that protein kinase C activation may enhance PTH degrading activity by UMR106 cells as a possible regulator of PTH degradation.     

CD40 signaling in B cells regulates the expression of the Pim-1 kinase via the NF-kappa B pathway.

The ability of CD40 signaling to regulate B cell growth, survival, differentiation, and Ig class switching involves many changes in gene expression. Using cDNA expression arrays and Northern blotting, we found that CD40 signaling increased the mRNA levels for pim-1, a protooncogene that encodes a serine/threonine protein kinase. Subsequent experiments showed that CD40 engagement also increased both Pim-1 protein levels and Pim-1 kinase activity in B cells. We then investigated the signaling pathways by which CD40 regulates Pim-1 expression and found that CD40 up-regulates Pim-1 primarily via the activation of NF-kappaB. Inhibiting the activation of NF-kappaB, either by treating cells with a chemical inhibitor, BAY11-7082, or by inducibly expressing a superrepressor form of IkappaBalpha, significantly impaired the ability of CD40 to increase Pim-1 protein levels. Because Pim-1 expression is associated with cell proliferation and survival, we asked whether this correlated with the ability of CD40 signaling to prevent anti-IgM-induced growth arrest in the WEHI-231 murine B cell line, a model for Ag-induced clonal deletion. We found that the anti-IgM-induced growth arrest in WEHI-231 cells correlated with a substantial decrease in Pim-1 levels. In contrast, culturing WEHI-231 cells with either anti-CD40 Abs or with the B cell mitogen LPS, both of which prevent the anti-IgM-induced growth arrest, also prevented the rapid decline in Pim-1 levels. This suggests that Pim-1 could regulate the survival and proliferation of B cells.     

Voltage-activated and stretch-activated Ba2+ conducting channels in an osteoblast-like cell line (UMR 106)

Calcium entry may mediate osteoblast activation by calciotropic hormones or changes in electrical fields or mechanical stress in bone. Using cell-attached patch clamping techniques, we have seen two Ba2+ conducting channels in UMR-106 osteoblast-like cells: (i) a voltage-dependent, dihydropyridine-sensitive channel resembling L-type Ca2+ channels in other cells, and (ii) a stretch-activated non-selective channel resembling those involved in mechanoreception and triggering of volume regulation in other cells.     

Angiotensin II induces NF-kappa B activation in HUVEC via the p38MAPK pathway

Angiotensin II (Ang II) is the main active peptide of the renin–angiotensin system (RAS), producing a number of inflammatory mediators that lead to endothelial dysfunction and the progression of atherosclerosis. Ang II-induced NF-κB nuclear translocation plays a pivotal role in this response. This study examines the NF-κB activation mechanism elicited by Ang II in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assays and Western blotting revealed that Ang II, signaling via AT₁, produces a time-dependent increase in NF-κB DNA binding and IκB degradation. These results also demonstrate that Ang II leads to MAPK phosphorylation and p38MAPK pathway-induced NF-κB activation. Furthermore, AT₁ is required for p38MAPK phosphorylation induced by Ang II. This study provides evidence that Ang II elicits NF-κB activation via the p38MAPK pathway in HUVEC.     

Calciotropic hormones raise the chemically detectable [Pi] in UMR 106–06 osteoblast-like cells

Uptake of orthophosphate (Pi) by osteoblast-like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast-like cells (UMR 106-06) that PTH (10^?11 to 10^?7 M ) increases both ³²Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D₃ (1,25D) (10^?12 to 10^?6 M ) and salmon calcitonin (CT) (10^?12 to 10^?6 g ml^?1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular ³²Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy- and 25 hydroxyvitamin D₃. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth.     

Heparin-binding epidermal growth factor-like growth factor inhibits cytokine-induced NF-kappa B activation and nitric oxide production via activation of the phosphatidylinositol 3-kinase pathway

NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-kappaB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-kappaB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by approximately 2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-kappaB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-kappaB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-kappaB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.     

Human osteoblast-like cells in three-dimensional culture with fluid flow

In a previous study, we showed that the combination of appropriately designed three-dimensional (3D) microcarrier scaffolds and fluid flow through and around the scaffolds during high aspect ratio vessel (HARV) rotation enhances the elaboration of mineralized bone matrix by osteoblast-like cells. In this study, we describe the ongoing characterization of our 3D culture system, including the investigation of interior fluid flow within the scaffolds and early stage integrin expression during hydrodynamic culture. Using theoretical and experimental methods, we have estimated that cells cultured on the interior of microcarrier scaffolds experience an interior nutrient flow velocity between 1 x 10(-3) and 1 x 10(-2) cm/s and maximum shear stress of 0.03 N/m(2). Under these conditions, osteoblast-like cells grew extensively in the interior regions of the scaffold and retained their osteoblastic phenotype as measured by alkaline phosphatase. In addition, flow cytometric analysis of the overall cell population showed that cells constitutively expressed integrin alpha3beta1 during 3D hydrodynamic culture.