Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK).

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38MAPK.

Substitution of two insulin receptor carboxy-terminal tyrosines with phenylalanine impairs the expression of MAP kinase phosphatase-1 (MKP-1) mRNA.

Cells expressing mutant insulin receptors (Y/F2), in which tyrosines 1316 and 1322 have been replaced with phenylalanine, exhibit enhanced insulin-induced MAP kinase activity and DNA synthesis in comparison with cells expressing wild type insulin receptors (Hirc B). To elucidate the mechanism of enhanced responsiveness, the expression of MAP kinase phosphatase-1 (MKP-1), a negative regulator of MAP kinase activity, was measured in Hirc B and Y/F2 cells incubated in the absence and presence of insulin for various periods of time, and over increasing concentrations of the ligand. Treatment of both cell lines with insulin induced a time and concentration-dependent relative increase in MKP-1 mRNA expression. However, in Y/F2 cells both basal and insulin-stimulated MKP-1 mRNA levels were more than 60% lower than that observed in cells transfected with the wildtype receptors. Cyclic AMP analog (8-Br-cAMP)/inducer (Forskoline) increased MKP-1 mRNA levels in both cell lines, and to a lesser extent in Y/F2 cells. In contrast to insulin the relative increase in MKP-1 mRNA expression induced by 8-Br-cAMP or forskoline was similar in Y/F2 and Hirc B cells. The overexpression of MKP-1 in Y/F2 cells inhibited insulin stimulated DNA synthesis. Transfection of wild type insulin receptors into Y/F2 cells increased basal levels of MKP-1. These results suggest that insulin receptor tyrosine residues 13/16 and 1322 play an important role in the regulation of MKP-1 expression both under basal and insulin stimulated conditions, and are not necessary for the induction of MKP-1 mRNA by cAMP. Furthermore, the enhanced insulin induced mitogenic signaling seen in Y/F2 cells is, at least in part, due to impaired MKP-1 expression.

Elevated expression and activity of protein-tyrosine phosphatase 1B in skeletal muscle of insulin-resistant type II diabetic Goto-Kakizaki rats.

We investigated the cellular mechanism(s) of insulin resistance associated with non-insulin dependent diabetes mellitus (NIDDM) using skeletal muscles isolated from non-obese, insulin resistant type II diabetic Goto-Kakizaki (GK) rats, a well known genetic rat model for type II diabetic humans. Relative to non-diabetic control rats (WKY), insulin-stimulated insulin receptor (IR) autophosphorylation and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation were significantly inhibited in GK skeletal muscles. This may be due to increased dephosphorylation by a protein tyrosine phosphatase (PTPase). Therefore, we measured skeletal muscle total PTPase and PTPase 1B activities in the skeletal muscles isolated from control rats (WKY) and diabetic Goto-Kakizaki (GK) rats. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate. Basal PTPase activity was 2-fold higher (P < 0.001) in skeletal muscle of GK rats when compared to WKY. Insulin infusion inhibited skeletal muscle PTPase activity in both control (26.20% of basal, P < 0.001) and GK (25.35% of basal, P < 0.001) rats. However, PTPase activity in skeletal muscle of insulin-stimulated GK rats was 200% higher than hormone-treated WKY controls (P < 0.001). Immunoprecipitation of PTPase 1B from skeletal muscle lysates and analysis of the enzyme activity in immunoprecipitates indicated that both basal and insulin-stimulated PTPase 1B activities were significantly higher (twofold, P < 0.001) in skeletal muscle of diabetic GK rats when compared to WKY controls. The increase in PTPase 1B activity in diabetic GK rats was associated with an increased expression of the PTPase 1B protein. We concluded that insulin resistance of GK rats is accompanied atleast by an abnormal regulation of PTPase 1B. Elevated PTPase 1B activity through enhanced tyrosine dephosphorylation of the insulin receptor and its substrates, may lead to impaired glucose tolerance and insulin resistance in GK rats.

Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction.

Insulin signaling involves a dynamic cascade of protein tyrosine phosphorylation and dephosphorylation. Most of our understanding of this process comes from studies focusing on tyrosine kinases, which are signal activators. Our knowledge of the role of protein-tyrosine phosphatases (PTPases), signal attenuators, in regulating insulin signal transduction remains rather limited. Protein-tyrosine phosphatase 1B (PTP-1B), the prototypical PTPase, is ubiquitously and abundantly expressed. Work from several laboratories, including our own, has implicated PTP-1B as a negative regulator of insulin action and as a potentially important mediator in the pathogenesis of insulin-resistance and non-insulin dependent diabetes mellitus (NIDDM).

Regulation of growth factor-induced signaling by protein-tyrosine-phosphatases.

The binding of a growth factor to its specific receptor catalyzes a complex cascade of intracellular signaling events, characterized by changes in the phosphorylation state of many key proteins. Among these phosphorylation events, tyrosine phosphorylation plays a prominent role in the transmission of postreceptor signals. The state of tyrosine phosphorylation is regulated by the actions of protein-tyrosine kinases (PTKs) and protein-tyrosine-phosphatases (PTPs). Dysregulation of either event can lead to abnormal cellular responses. PTPs generally act to regulate negatively-that is, to turn off-any signals generated by PTKs. However, this is not always the case, as seen by the phosphatase SHP-2, which can either be a positive or negative regulator of signal transduction depending on the particular cellular context. In addition, a novel family of dual specificity phosphatases has been recently discovered. These enzymes are capable of dephosphorylating phosphotyrosine and phosphothreonine/phosphoserine residues, and seem to play a significant role in attenuating the action of MAP kinases. Several themes appear throughout PTP regulation of growth factor signaling, including positive or negative regulation, importance of cell/ tissue type, identity of the receptor activated, and subcellular localization. Although only a handful of PTPs have been identified, the present work done in elucidating their function has revealed their significance in the maintenance of normal physiological responses to growth factors.

Insulin-induced mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) attenuates insulin-stimulated MAP kinase activity: a mechanism for the feedback inhibition of insulin signaling.

Insulin signaling involves the transient activation/inactivation of various proteins by a cycle of phosphorylation/dephosphorylation. This dynamic process is regulated by the action of protein kinases and protein phosphatases. One family of protein kinases that is important in insulin signaling is the mitogen-activated protein (MAP) kinases, whose action is reversed by specific MAP kinase phosphatases (MKPs). Insulin stimulation of Hirc B cells overexpressing the human insulin receptor resulted in increased MKP-1 mRNA levels. MKP-1 mRNA increased in a dose-dependent manner to a maximum of 3- to 4-fold over basal levels within 30 min, followed by a gradual return to basal. The mRNA induction did not require the continuous presence of insulin. The induction of MKP-1 protein synthesis followed MKP-1 mRNA induction; MKP-1 protein was maximally expressed after 120 min of insulin stimulation. MKP-1 mRNA induction by insulin required insulin receptor tyrosine kinase activity, since overexpression of an altered insulin receptor with impaired intrinsic tyrosine kinase activity prevented mRNA induction. Forskolin, (Bu)2-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP increased the MKP-1 mRNA content moderately above basal. These agents also augmented the insulin-stimulated expression of MKP-1 mRNA. However, in some cases the increase in MKP-1 mRNA expression was less than additive. Nevertheless, these results indicate that multiple signaling motifs might regulate MKP-1 expression and suggest another mechanism for the attenuation of insulin-stimulated MAP kinase activity by cAMP. Overexpression of MKP-1 in Hirc B cells inhibited both insulin-stimulated MAP kinase activity and MAP kinase-dependent gene transcription. The results of these studies led us to conclude that insulin regulates MKP-1 and strongly suggest that MKP-1 acts as a negative regulator of insulin signaling.

Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor.

This study examined the effects of leukemia inhibitory factor (LIF) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). LIF induced a dose-dependent increase in p24 antigen production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the LIF effects were similar to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these LIF effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. LIF increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The DNA binding protein NF-kB is a central mediator in cytokine activation of HIV transcription. NF-kB levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines LIF increased NF-kB activity. Induction of NF-kB and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of nitric oxide synthase, inhibited LIF-induced HIV replication. LIF induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to IL-1, TNF, or IL-6. These results identify LIF as a stimulus of HIV replication.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-1 beta and IL-6 selectively induce transforming growth factor-beta isoforms in human articular chondrocytes.

Transforming growth factor-beta (TGF-beta) plays an important role in homeostasis of connective tissues, but regulation of its expression in mesenchymal cells is not well characterized. This study examines the effects of the cytokines IL-1 beta and IL-6 on expression of TGF-beta isoforms in human articular chondrocytes. IL-6 caused a fivefold increase, in the secretion of TGF-beta bioactivity by primary chondrocytes, whereas IL-1 beta showed only marginal stimulatory effects. Analysis by Northern blotting showed that IL-6 induced TGF-beta 1 gene expression but had no detectable effect on TGF-beta 2 mRNA levels and marginally increased TGF-beta 3 mRNA. However, IL-1 inhibited TGF-beta 1 mRNA expression induced by serum. In contrast, IL-1 beta strongly and selectively upregulated the TGF-beta 3 isoform. To determine whether this differential effect of IL-1 beta resulted in a corresponding change in protein synthesis, chondrocytes were metabolically labeled and analyzed by immunoprecipitation. IL-1 beta selectively induced TGF-beta 3 protein synthesis but reduced synthesis of the TGF-beta 1 and TGF-beta 2 isoforms. Consistent with the effects on TGF-beta 1 mRNA, IL-6 increased the synthesis of TGF-beta 1. These differential effects of the cytokines IL-1 beta and IL-6 provide new insight into the regulation of TGF-beta expression and may represent a protective mechanism against cytokine-induced connective tissue catabolism.