Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin.

Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization.     

Role of the β1 integrin molecule in T-cell activation and migration

β1 integrins play crucial roles in a variety of cell processes such as adhesion, migration, proliferation, and differentiation of lymphocytes. To understand the molecular mechanisms of these various biological effects, it is particularly important to analyze cell signaling through the β1 integrins. Our previous study showed that PLC-γ, pp125FAK (focal adhesion kinase), pp105, paxillin, p59fyn, p56lck, and ERK1/2 are phosphorylated in their tyrosine residues upon engagement of β1 integrins. We identified pp105 as Cas (Crk-associated substrate)-related protein and successfully cloned its cDNA. pp105 is a Cas homologue predominantly expressed in the cells of lymphoid lineage, which led us to designate it Cas-L. Like p130Cas, Cas-L contains a single SH3 domain and multiple SH2-binding sites (YXXP motif), which are suggested to bind SH2 domains of Crk, Nck, and SHPTP2. Subsequent studies revealed that pp125FAK binds Cas-L on its SH3 domain and phosphorylates its tyrosine residues upon β1 integrin stimulation. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. We have shown that Cas-L is involved in the T-cell receptor (TCR)/CD3 signaling pathway as well as the β1 integrin signaling pathway. Cas-L is transiently phosphorylated following CD3 crosslinking and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant (Cas-LΔSH3), which lacks the binding site for FAK, is still tyrosine-phosphorylated upon CD3 crosslinking but not upon β1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Finally, we have identified a crucial role of Cas-L in β1 integrin-mediated T-cell co-stimulation. We have found that this co-stimulatory pathway is impaired in the Jurkat T-cell line, and that the expression level of Cas-L is reduced in the Jurkat cells compared to peripheral T-cells. The transfection of Cas-L cDNA into Jurkat cells restored the β1 integrin-mediated co-stimulation, while the transfection of Cas-LΔSH3 mutant failed to do so, which contrasts with the case of CD3-mediated signaling. These results indicate that Cas-L plays a key role, through the association and phosphorylation by FAK, in β1 integrin-mediated T-cell co-stimulation. Moreover, tyrosine phosphorylation of Cas-L is critical for T-cell receptor and β1 integrin-induced T-lymphocyte migration. Taken together, Cas-L might be the bi-modal docking protein which assembles the signals through β1 integrins and TCR/CD3, and which participates in a variety of T-cell functions. Received: August 24, 1999 / Accepted: August 31, 1999     

Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.     

PTEN and SHIP2 regulates PI3K/Akt pathway through focal adhesion kinase

Our laboratory has established a novel role of focal adhesion kinase (FAK) in vitro and in vivo, as a positive regulator of insulin signaling pathway. In vitro studies reported tyrosine dephosphorylation of FAK under insulin resistance in C2C12 skeletal muscle cells. A decrease in FAK tyrosine phosphorylation was also observed in skeletal muscle of insulin resistant Sprague–Dawley rats fed on high-fat-diet. Present study was undertaken to explore the cellular mechanism of FAK dephosphorylation under insulin resistance in C2C12 skeletal muscle cells. Here we report that PTEN and SHIP2, the phosphatases widely implicated as negative regulators of insulin signaling, to be responsible for dephosphorylation of FAK. Data propose that under insulin resistance upregulation of PTEN and SHIP2 act through changes in FAK phosphorylation to impair insulin signaling suggesting FAK to be a key mediator of PTEN and SHIP2 in the regulation of insulin signaling. Thus data elucidates a part of molecular mechanism of insulin resistance in skeletal muscle cells.     

Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras.

Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The pY542TNI sequence conforms to the consensus binding site for the SH2 domain of Grb2, which, by association with Sos1, couples some growth factor receptors to Ras. Following PDGF stimulation, Grb2 binds tyrosine-phosphorylated SHPTP2. Moreover, a mutant PDGFR lacking its SHPTP2 binding site displays markedly reduced Grb2 binding. These data indicate that phosphorylation of SHPTP2 couples Grb2 to PDGFR in vivo, providing a mechanism for Ras activation by PDGFR and for positive signaling via SHPTP2 and Csw.     

Lack of SHPTP1 results in src-family kinase hyperactivation and thymocyte hyperresponsiveness.

Protein tyrosine phosphorylation and dephosphorylation are key regulatory events in T-cell receptor (TCR) signaling. We investigated the role of the tyrosine phosphatase SHPTP1 in TCR signaling by analysis of TCR signal transduction in motheaten (me/me) mice, which lack SHPTP1 expression. As revealed by flow cytometric analysis, thymocyte development was normal in me/me mice. However, me/me thymocytes hyperproliferated (3-to 5-fold) in response to TCR stimulation, whereas their response to interleukin 2 stimulation was unchanged compared with normal thymocytes. TCR-induced hyperproliferation of me/me thymocytes was reproduced in purified single-positive thymocytes. Moreover, me/me thymocytes produced increased amounts of interleukin 2 production upon TCR stimulation. Biochemical analysis revealed that, in response to TCR or TCR/CD4 stimulation, thymocytes lacking SHPTP1 showed increased tyrosyl phosphorylation of several cellular substrates, which correlated with increased activation of the src-family kinases Lck and Fyn. Taken together, our data suggest that SHPTP1 is an important negative regulator of TCR signaling, acting at least in part to inactivate Lck and Fyn.     

Aurintricarboxylic acid induces a distinct activation of the IGF-I receptor signaling within MDA-231 cells

Aurintricarboxylic acid (ATA), a polymeric carboxylated triphenylmethane derivate, prevents apoptotic death in a variety of cell systems. Recently, we have shown that the survival promoting effect of ATA is transduced via activation of the IGF-I receptor (IGF-IR) signaling pathway. In breast cancer MDA-231 cells exposed either to the protein synthesis inhibitors cycloheximide or ricin or to the anticancer drug adriamycin, we have found that ATA, but not IGF-1, is a powerful antiapoptotic agent. The purpose of this study was to compare the ability of ATA and IGF-I to activate the IGF-IR signaling cascade and to correlate this ability to their survival potency. MDA-231 cells were exposed to ATA or IGF-I, up to 7 h, and the dynamics of activation of the IGF-IR signaling cascade was evaluated. Our results show that: 1) The amount of tyrosine phosphorylated IGF-IR proteins was greater after exposure to ATA, compared with IGF-I. 2) Two phosphorylated IGF-IR beta-subunits (a 95-kDa and a 75-kDa) were induced after exposure to ATA, whereas IGF-1 induced only the 95-kDa form. Immunoprecipitation of both receptor forms by antibodies against the alpha-subunit and against the carboxy terminus of the beta-subunit of the IGF-IR suggests that the 75-kDa form could be the beta-chain truncated at the amino terminus above the alpha-beta disulphide bridges. 3) The ATA-activated IGF-IR forms underwent slow dephosphorylation, compared with a rapid dephosphorylation of the IGF-I activated receptor. 4) The insulin receptor substrate-1/2-associated PI3K, Shc proteins, and the kinases Akt and Erk1/2, downstream mediators of the antiapoptotic signaling by IGF-IR, were activated to a higher extent and for a longer time period by ATA, compared with IGF-I. Taken together, the sustained activation of the IGF-IR signaling pathway by ATA may explain its stronger antiapoptotic effect. We suggest that this enhanced activity, and the different susceptibility of the IGF-IR to certain proteases and phosphatases, may indicate a distinct conformation of the ATA-activated IGF-IR.     

Reduced expression of focal adhesion kinase disrupts insulin action in skeletal muscle cells

Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-(14)C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr(397) phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.     

Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.

Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix.     

Nucleobindin-2 is a positive modulator of EGF-dependent signals leading to enhancement of cell growth and suppression of adipocyte differentiation

Nucleobindin-2 is a 420-amino-acid EF-hand calcium-binding protein that undergoes proteolytic processing to generate an 82-amino-acid amino-terminal peptide termed nesfatin-1. To determine whether nucleobindin-2 has any biological function, nucleobindin-2 was either overexpressed or knocked down by short hairpin RNA in cultured CHO cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IE) and in 3T3-L1 cells. Reduction in nucleobindin-2 expression inhibited EGF-stimulated MAPK kinase (S217/S221) and Erk phosphorylation (T202/Y204). In contrast, there was no significant effect on EGF-stimulated EGF receptor phosphorylation, EGF receptor internalization, or 52-kDa Shc and c-Raf phosphorylation. Although kinase suppressor of Ras-1 and protein phosphatase 2A expression was not changed, intracellular calcium concentrations and PP2A activity was significantly increased in nucleobindin-2 knocked-down cells. Concomitant with these alterations in EGF-stimulated signaling, cell proliferation was significantly reduced in nucleobindin-2 knocked-down cells. Moreover, reduced nucleobindin-2 expression in 3T3-L1 preadipocytes resulted in a greater extent of 3T3-L1 cell adipocyte differentiation. Taken together, these data indicate that nucleobindin-2 regulates EGF-stimulated MAPK kinase/Erk signaling, cell proliferation, and adipocyte differentiation.     

GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis.     

Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors.

Porcine and bovine GH receptor (GHR) cDNAs werestably expressed in mouse L cells, which normally do not possess detectablelevels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of125I-labeled GH by these cell lines. To study GHR-related signaling events inthese cells, protein tyrosine phosphorylation was examined. In GH-treated cells,a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa(pp95) was increased dramatically (approximately 100-fold) relative tonon-GH-treated cells. The amount of pp95 within the cells after GH treatment waspositively correlated with the number of GHRs on the cells. Tyrosinephosphorylation of pp95 could not be induced by prolactin, insulin, insulin-likegrowth factor I, interleukin 2, epidermal growth factor, platelet-derived growthfactor, or fibroblast growth factor. Phosphorylation of pp95 was found to be arapid event that could be observed 60 sec after GH treatment. Also, pp95 appearsto exist as a complex of two proteins, i.e., pp95 and pp96. The GH-inducedresponse by these cells may be of use in screening GH analogs for biologicalactivity.     

Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino acids, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpression of RAFTK and FAK proteins in megakaryocytic cells and blood platelets was observed. Using a specific antibody to RAFTK and the monoclonal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigenically. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells transfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Similar to FAK, dephosphorylation of RAFTK was observed when adherent transfected COS cells were detached. Phosphorylation was regained upon replating of these cells on the fibronectincoated dishes. Analysis of tyrosine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinases as well as the Grb2 adaptor protein were able to specifically associate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was observed upon fibronectin-induced activation of human megakaryocytic cells. Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesion- like structures in adherent CMK cells and in transfected pCDNAIII/flag- RAFTK COS cells upon fibronectin activation. These data suggest that RAFTK is a novel member of the FAK family, that it localizes to focal adhesion-like structures in CMK megakaryocytic cells, that it participates in integrinmediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions.     

Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function.

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.     

Regulation of PAF-induced platelet responses by cyclic nucleotides

The existence of cross-talk mechanisms between the cyclic nucleotide system and other transduction systems involved in PAF-activated platelets is described in this study. A protein of 125 kDa, identified as pp125FAK, is tyrosine phosphorylated by PAF in a time- and concentration-dependent manner. The presence of a cAMP- or a cGMP-elevating agent, used alone or in combination, together with PAF diminished tyrosine phosphorylation. The sensitivity to cAMP shown by PAF-induced pp125 phosphorylation on tyrosine residues was similar to PAF-induced phosphorylation of a 47-kDa protein (pp47) on serine and threonine. In contrast, the latter was not affected in the presence of a cGMP-elevating agent, although it was able to enhance synergistically the inhibitory effect of forskolin. Data reported herein also show that pp47 phosphorylation and serotonin secretion are not closely correlated. Accordingly, sodium nitroprusside (SNP) did not have any effect on phosphorylation of pp47, but it was able to inhibit serotonin secretion when added alone, and it showed a synergistic inhibitory action with forskolin.     

Lymphocyte antigen receptor activation of a focal adhesion kinase-related tyrosine kinase substrate.

One of the earliest responses of T and B lymphocytes to stimulation through their antigen receptors is the activation of protein tyrosine kinases and the tyrosine phosphorylation of multiple cellular substrates. Here we describe a tyrosine kinase substrate, fakB, a putative homologue of the focal adhesion kinase pp125FAK. Tyrosine phosphorylation of fakB was rapidly augmented in human T and B cells following antigen receptor cross-linking with antibody, while pp125FAK was nonresponsive. Costimulation of the T-cell antigen receptor (TCR/CD3) with either the CD2 or CD4 costimulatory receptors induced synergistic fakB tyrosine phosphorylation in normal human T cells. Engagement of TCR/CD3 induced the stable association of fakB with ZAP-70, the TCR/CD3 sigma-chain-associated tyrosine kinase involved in antigen receptor-induced T-cell activation. In addition, preformed complexes of fakB and ZAP-70 were observed in T-cell leukemia lines. Phosphorylation of fakB on serine, threonine, and tyrosine residues was observed both in vivo and in vitro, where a functional increase of in vitro kinase activity was observed following TCR/CD3 stimulation. fakB is thus a focal adhesion kinase-related tyrosine kinase substrate that is differentially regulated from that of pp125FAK and likely plays a role in antigen-induced lymphocyte signaling.     

The HIV protease inhibitor indinavir impairs glycogen synthesis in HepG2 hepatoma cells.

HIV protease inhibitor treatment is associated with insulin resistance. We have recently demonstrated that the HIV protease inhibitor indinavir influences initial insulin signaling steps in HepG2 cells. Here we investigated in the same cell model whether indinavir alters insulin-stimulated glycogen synthesis. Since an altered phosphotyrosine phosphatase activity could represent a mechanism by which insulin signaling is influenced, we also assessed potential indinavir effects on protein tyrosine phosphatase activity directed against tyrosine phosphorylated insulin receptor substrate-1. HepG2 cells were incubated for 48 h without or with indinavir (100 micro mol/l). Subsequently, the insulin-stimulated incorporation of 14C-glucose into glycogen was measured. In indinavir-treated cells the insulin effect on glycogen synthesis was reduced by 30 +/- 4.5 %. Dephosphorylation of immobilized tyrosine-phosphorylated insulin-receptor substrate-1 by the cell extracts was determined using a microwell plate-based method, and indinavir treatment did not alter this dephosphorylation. In conclusion, our data suggest that indinavir affects insulin-stimulation of glycogen synthesis in liver cells, and this may be related to the previously observed alterations in insulin signaling. Direct effects of indinavir on the GLUT4 transport system, that have been suggested from data in other cell systems, are unlikely in HepG2 cells that express no or almost no GLUT4 transport system. Finally, our data do not support the hypothesis that indinavir alters insulin signaling by influencing protein tyrosine phosphatase activity directed against insulin receptor substrate-1.     

Proline-rich tyrosine kinase 2 and Src kinase signaling transduce monosodium urate crystal-induced nitric oxide production and matrix metalloproteinase 3 expression in chondrocytes

OBJECTIVE: Articular deposition of monosodium urate monohydrate (MSU) crystals may promote cartilage and bone erosion. Therefore, the aim of this study was to determine how MSU crystals stimulate chondrocytes. METHODS: Nitric oxide (NO) release, and expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 3 (MMP-3) were assessed in cultured chondrocytes treated with MSU. MSU-induced functional signaling by specific protein kinases (p38, Src, and the focal adhesion kinase [FAK] family members proline-rich tyrosine kinase 2 [Pyk-2] and FAK) was also examined using selective pharmacologic inhibitors and transfection of kinase mutants. RESULTS: MSU induced MMP-3 and iNOS expression and NO release in chondrocytes in a p38-dependent manner that did not require interleukin-1 (IL-1), as demonstrated by using IL-1 receptor antagonist. MSU induced rapid tyrosine phosphorylation of Pyk-2 and FAK, their adaptor protein paxillin, and interacting kinase c-Src. Pyk-2 and c-Src signaling both mediated p38 MAPK activation in response to MSU. Pyk-2 and c-Src signaling played a major role in transducing MSU-induced NO production and MMP-3 expression. But, despite the observed FAK phosphorylation, a selective pharmacologic FAK inhibitor and a FAK dominant-negative mutant both failed to block MSU-induced NO release or MMP-3 expression in parallel experiments. CONCLUSION: In chondrocytes, MSU crystals activate a signaling kinase cascade typically employed by adhesion receptors that involves upstream Src and FAK family activation and downstream p38 activation. In this cascade, Pyk-2, Src, and p38 kinases transduce MSU-induced NO production and MMP-3 expression. Our results identify Pyk-2 and c-Src as novel sites for potential therapeutic intervention in cartilage degradation in chronic gout.     

Targeting focal adhesion kinase with small interfering RNA prevents and reverses load-induced cardiac hypertrophy in mice

Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing ( approximately 70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of beta-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.     

Proline‐rich tyrosine kinase 2 and Src kinase signaling transduce monosodium urate crystal–induced nitric oxide production and matrix metalloproteinase 3 expression in chondrocytes

Objective

Articular deposition of monosodium urate monohydrate (MSU) crystals may promote cartilage and bone erosion. Therefore, the aim of this study was to determine how MSU crystals stimulate chondrocytes.

Methods

Nitric oxide (NO) release, and expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 3 (MMP‐3) were assessed in cultured chondrocytes treated with MSU. MSU‐induced functional signaling by specific protein kinases (p38, Src, and the focal adhesion kinase [FAK] family members proline‐rich tyrosine kinase 2 [Pyk‐2] and FAK) was also examined using selective pharmacologic inhibitors and transfection of kinase mutants.

Results

MSU induced MMP‐3 and iNOS expression and NO release in chondrocytes in a p38‐dependent manner that did not require interleukin‐1 (IL‐1), as demonstrated by using IL‐1 receptor antagonist. MSU induced rapid tyrosine phosphorylation of Pyk‐2 and FAK, their adaptor protein paxillin, and interacting kinase c‐Src. Pyk‐2 and c‐Src signaling both mediated p38 MAPK activation in response to MSU. Pyk‐2 and c‐Src signaling played a major role in transducing MSU‐induced NO production and MMP‐3 expression. But, despite the observed FAK phosphorylation, a selective pharmacologic FAK inhibitor and a FAK dominant‐negative mutant both failed to block MSU‐induced NO release or MMP‐3 expression in parallel experiments.

Conclusion

In chondrocytes, MSU crystals activate a signaling kinase cascade typically employed by adhesion receptors that involves upstream Src and FAK family activation and downstream p38 activation. In this cascade, Pyk‐2, Src, and p38 kinases transduce MSU‐induced NO production and MMP‐3 expression. Our results identify Pyk‐2 and c‐Src as novel sites for potential therapeutic intervention in cartilage degradation in chronic gout.