Extracellular calcium induces COX-2 in osteoblasts via a PKA pathway

We have shown that extracellular calcium [Ca(+2)](e) induces cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production via an ERK signaling pathway in osteoblasts. In this study, we examined the roles of protein kinase C (PKC) and A (PKA) signaling pathways in the [Ca(+2)](e) induction of COX-2 in primary calvarial osteoblasts from mice transgenic for -371 bp of the COX-2 promoter fused to a luciferase reporter. Neither PKC specific inhibitors nor downregulation of the PKC pathway by phorbol myristate acetate (PMA) affected the [Ca(+2)](e) stimulation of COX-2 mRNA or promoter activity. In contrast, PKA inhibitors, used at doses that inhibited forskolin-stimulated luciferase activity by 90%, reduced [Ca(+2)](e)-stimulated COX-2 mRNA expression and promoter activity by 80-90%. [Ca(+2)](e) also stimulated a 2- to 3-fold increase in cAMP production. Hence, the [Ca(+2)](e) induction of COX-2 mRNA expression and promoter activity was independent of the PKC pathway and dependent on the PKA signaling pathway.     

Transforming growth factor-beta1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways

Transforming growth factor beta 1 (TGF-beta1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-beta1; if the physiological response occurs via type II or type III TGF-beta receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-beta1 stimulated [(3)H]thymidine and [(35)S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-beta1-dependent PKC and the physiological response of GC cells to TGF-beta1 was reversed by anti-type II TGF-beta receptor antibody and soluble type II TGF-beta receptor, showing that TGF-beta1 mediates these effects through the type II receptor. The increase in [3H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-beta1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-beta1 activation of PKC is through phospholipase A(2) (PLA(2)) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-beta1. Prostanoids are required, as indomethacin blocked the effect of TGF-beta1, and Cox-1, but not Cox-2, is involved. TGF-beta1 stimulates prostaglandin E(2) (PGE(2)) production and exogenous PGE(2) stimulates PKC, but not as much as TGF-beta1, suggesting that PGE(2) is not sufficient for all of the prostaglandin effect. In contrast, TGF-beta1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-beta1 signaling at different levels in the cascade. TGF-beta1-dependent increases in PGE(2) levels and PKC were augmented by the G protein activator GTP gamma S, whereas inhibition of G-protein activity via GDP beta S, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-beta1, indicating that both G(i) and G(s) are involved.Inhibition of PKA with H-8 partially blocked TGF-beta1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-beta1 stimulates PLA(2) and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE(2) activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.     

HIV-1 Tat protein induces IL-10 production by an alternative TNF-alpha-independent pathway in monocytes: role of PKC-delta and p38 MAP kinase

HIV-1 Tat protein stimulates the production of both TNF-alpha and IL-10 in human monocytes. Taking into account the ability of TNF-alpha to induce IL-10 production, we evaluated the link between Tat, TNF-alpha and IL-10 and the implication of PKC and p38 MAP kinase pathways. Our data showed that (i) in the presence of neutralizing anti-TNF-alpha antibodies, IL-10 production is only partially inhibited; (ii) in a calcium-free medium, while TNF-alpha production is totally inhibited, Tat continues to induce IL-10; (iii) under these conditions, Tat-mediated IL-10 production is associated with PKC-delta activation; and (iv) downstream of PKC, p38 MAP kinase is crucial for TNF-alpha independent IL10 production. Overall, our data suggest a new mechanism, implicating Tat protein, by which HIV-1 may maintain a constant production of the immunosuppressive IL-10 cytokine, even in the absence of TNF-alpha production. In consequence, HIV-1 may escape immune surveillance and thus promote the establishment of an immunosuppressive state.     

Release of calcium from inositol 1,4,5-trisphosphate receptor-regulated stores by HIV-1 Tat regulates TNF-alpha production in human macrophages

HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-alpha, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-alpha production in macrophages, and we have shown that Tat releases calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-alpha production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-alpha production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-alpha production.     

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.     

Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus

Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-alpha, IL-15, IL-12) cytokines, hsp27, a small hsp, has been primarily identified as a substrate of mitogen-activated protein kinase-activated protein kinase-2 involved in the p38 signaling pathway and activated during monocyte IL-10 production. Hsp27 can also act as an endogenous protein circulating in the serum of breast cancer patients and a protein whose induction correlates to protection from LPS shock. However, the cytokine-stimulating properties of hsp27 have been unexplored. In this study, exogenous hsp27 is demonstrated for the first time as a potent activator of human monocyte IL-10 production, but only a modest inducer of TNF-alpha. Although exogenous hsp27 stimulation activated all three monocyte mitogen-activated protein kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38), only p38 activation was sustained and required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and p38 activation were required for induction of TNF-alpha when using the p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27's transient activation of the c-Jun N-terminal kinase pathway, which can down-regulate IL-10, may contribute to its potent IL-10 induction. Hsp27's ERK 1/2 activation was also less sustained than activation by stimuli like LPS, possibly contributing to its modest TNF-alpha induction. The failure of either PD98059 or anti-TNF-alpha Ab to substantially inhibit IL-10 induction implied that hsp27 induces IL-10 via activation of p38 signaling independently of TNF-alpha activation and may be predominantly an antiinflammatory monokine stimulus.     

HIV-1 Tat protein induces IL-10 production in monocytes by classical and alternative NF-kappaB pathways

The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-kappaB-dependent pathways. The trans-dominant negative mutants of NF-kappaB-inducing kinase (NIK), IKKalpha and IKKbeta expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-kappaB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-kappaB pathways. In inactivated cells, IKKalpha is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKalpha translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKalpha and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-kappaB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKalpha nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKalpha pathways, to promote production of IL-10.     

Regulation of interleukin 6 production in a human gastric epithelial cell line MKN-28

Interleukin (IL-) 6 is closely related to gastrointestinal diseases. The question of whether gastric epithelial cell contributes to IL-6 production remains undefined. We aim to evaluate the regulatory pathway of IL-6 expression in gastric epithelial cells, by using different inflammatory cytokines, endotoxin, or protein kinase modulators. IL-6 was measured by ELISA. Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187, TNF-alpha, IL-1beta, oncostatin M (OSM) but not lipopolysaccharide stimulated IL-6 production from gastric epithelial cell line MKN-28. Blocking protein tyrosine kinase (PTK) activation by herbimycin A or genistein, or blocking NF-kappaB activation by pyrrolidinedithiocarbamate, reduced the IL-6 expression induced by TNF-alpha, IL-1beta and OSM. Dexamethasone mimicked this effect. Protein kinase (PK) C inhibitor only reduced the PMA and OSM induced IL-6 production. Both inhibitors and activators for PKA and G-protein as well as IL-10 had no effects on IL-6 expression. These results indicate that inflammatory cytokines are crucial for IL-6 regulation in gastric epithelial cells. The IL-6 signal pathway is mediated through PTK, NF-kappaB, and also involve PKC, intracellular calcium and sensitive to dexamethasone, but is not related to PKA, G-protein and IL-10.     

Regulation of expression of granulocyte-macrophage colony-stimulating factor in human bronchial epithelial cells: roles of protein kinase C and mitogen-activated protein kinases

GM-CSF has a major role in the immune and inflammatory milieu of the airway. Airway epithelial cells (AEC) are among the first targets of environmental stimuli and local cytokines, in response to which they can produce GM-CSF. The regulation of GM-CSF is only minimally understood in AEC. We hypothesized that GM-CSF expression in AEC would result from activation of protein kinase C (PKC) and subsequent activation of the extracellular signal-regulated kinase (MAPKerk1/2) pathway, so we investigated signal transduction pathways in human primary culture bronchial epithelial cells (HBECs). TNF-alpha, IL-1beta, and PMA induced the release of GM-CSF in HBECs. The robust response to PMA was not detected in SV40 adenovirus-transformed normal human bronchial epithelial cells (BEAS-2B). PMA and TNF-alpha stimulation of GM-CSF required activation of PKC (inhibition by staurosporine and bisindolylmaleimide I). GM-CSF expression was up-regulated by a nonphorbol PKC activator, but not by an inactive PMA analogue. PMA-induced GM-CSF production in HBECs did not require a Ca2+ ionophore and was not inhibited by cyclosporin A. Activation of MAPKerk1/2 via PKC was associated with and was required for GM-CSF production induced by PMA and TNF-alpha. The data demonstrate regulation of GM-CSF in HBECs by PKC pathways converging on the MAPKerk1/2 pathway and further define cell-specific regulation critical for local airway responses.     

Globular adiponectin decreases leptin-induced tumor necrosis factor-alpha expression by murine macrophages: involvement of cAMP-PKA and MAPK pathways

Several lines of evidence have supported a link between obesity and inflammation. The present study investigated the capacity of leptin and globular adiponectin to affect tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. Leptin stimulated TNF-alpha production at mRNA as well as protein levels in a dose- and time-dependent manner. Intracellular cAMP concentration was increased and protein kinase A (PKA) was activated with the treatment of leptin, subsequently downstream MAPK signal proteins, ERK1/2 and p38, were phosphorylated. Specific inhibitors for the signal proteins, Rp cAMPS, H89, PD98059, and U0126, or SB203580, suppressed the signaling pathway and TNF-alpha expression. Although gAd partially increased cAMP concentration and PKA activity, it directly reduced leptin-induced ERK1/2 and p38 MAPK phosphorylation thus inhibiting TNF-alpha production. In conclusion, leptin promotes inflammation by stimulating TNF-alpha production, which is mediated by cAMP-PKA-ERK1/2 and p38 MAPK pathways. gAd inhibited leptin-induced TNF-alpha production through suppressing phosphorylation of ERK1/2 and p38 pathways.     

Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor-alpha (TNF-alpha ) signal transduction in L929 cells. Translocation and inactivation of PKC by TNF-alpha

Tumor necrosis factor-alpha (TNF-alpha) is capable of inducing a variety of biologic responses through multiple signaling pathways. Because of the potential role of protein kinase C (PKC) in apoptosis, we examined the effects and mechanisms of TNF-alpha on PKC regulation, specifically on PKC alpha. In L929 murine fibroblasts, TNF-alpha (0.5- 5 nm) caused potent inhibition of PKC alpha activity and induced translocation of PKC alpha from the cytosol to the membrane. Treatment of cells with TNF-alpha also induced dephosphorylation of PKC alpha as detected by a mobility shift on SDS-polyacrylamide gel and inhibition of PKC phosphorylation as probed by anti-phospho-PKC antibodies. Since PKC is activated directly by diacylglycerol and inactivated indirectly by ceramide, we next examined the roles of these lipid mediators in the regulation of PKC alpha. Addition of TNF-alpha led to accumulation of both ceramide and diacylglycerol. Fumonisin B(1), an inhibitor of ceramide synthase, and glutathione, an inhibitor of neutral sphingomyelinase, both reversed the effect of TNF-alpha on PKC alpha activity, suggesting that ceramide production is necessary for the action of TNF-alpha. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKC alpha, but not the mobility shift. Okadaic acid at 2 nm, a potent protein phosphatase inhibitor, blocked the effects of TNF-alpha on PKC alpha activity, but not on PKC alpha translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKC alpha acts as a downstream target for TNF-alpha and that different lipid-mediated pathways in TNF-alpha signaling lead to opposing signals in the regulation of PKC alpha activity.     

Cordyceps sinensis mycelium activates PKA and PKC signal pathways to stimulate steroidogenesis in MA-10 mouse Leydig tumor cells

Cordyceps sinensis (CS) mycelium stimulates steroidogenesis in MA-10 mouse Leydig tumor cells, but the mechanisms remain unclear. In this study, MA-10 cells were treated with different reagents in the presence or absence of CS (10 mg/ml) for 3 h to determine the mechanisms. Results illustrated that CS activated the Gsalpha protein subunit, but not Gialpha, to induce cell steroidogenesis. Moreover, PKA inhibitors inhibited 37% of CS-stimulated steroidogenesis, which demonstrated that CS might enhance the cAMP-PKA pathway to affect MA-10 cell steroidogenesis. Because of incomplete inhibition by PKA inhibitors, we also examined the PKC pathway. PKC inhibitor, phospholipase C inhibitor, and calmodulin antagonist blocked 35-52% of CS-stimulated steroidogenesis in MA-10 cells, strongly suggesting that CS had activated the PKC pathway. Co-treatment with PKA and PKC inhibitors abolished 61% of CS-stimulated steroid production, indicating that CS simultaneously activated PKA and PKC pathways. Moreover, CS induced the expression of steroidogenic acute regulatory (StAR) protein in dose- and time-dependent relationships, and PKA inhibitor, PKC inhibitor, or co-treatment with both inhibitors suppressed it. These data support that CS activates both PKA and PKC signal transduction pathways to stimulate MA-10 cell steroidogenesis.     

alpha-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production in leukocytes by modulating protein kinase A,p38 kinase,and nuclear factor kappa B signaling pathways

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in leukocytes via stimulation of alpha-MSH cell surface receptors. However, the signaling mechanism of alpha-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which alpha-MSH inhibits lipopolysaccharide (LPS)-induced TNF-alpha production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor kappa B (NF kappa B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF kappa B to induce TNF-alpha production. We found that pretreatment of cells with alpha-MSH blocked LPS-induced p38 kinase and NF kappa B activation as well as TNF-alpha production. This response was proportional to alpha-MSH receptor expression levels, and addition of an alpha-MSH receptor antagonist abolished the inhibitory effects. In addition, alpha-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of alpha-MSH on p38 kinase activation, NF kappa B activation, and TNF-alpha production. Taken together, our results indicate that stimulation of PKA by alpha-MSH causes inhibition of LPS-induced activation of p38 kinase and NF kappa B to block TNF-alpha production.     

Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C

Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH-stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH-treated) luteal cells had an additive effect on progesterone production. In TSH-treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum.