The mitogen-activated protein (MAP) kinases and ribosomal S6 protein kinases in the skeletal muscle of insulin-resistant long-term (2 and 6 months' duration) diabetic rats were investigated to understand further the changes in insulin intracellular signaling pathways that accompany diabetes. The effects of insulin-mimetic vanadium compounds on the activity of these kinases were also examined. In the insulin-resistant 2-month diabetic rats, the basal activities of MAP kinases were relatively unchanged, while the basal activities of S6 kinases were significantly increased. Intravenous injection of insulin moderately activated both the 42-kDa MAP kinase (p42mapk) and a 44-kDa MAP kinase (p44erk1) in the 2-month control rats but not in the 2-month diabetic rats. Insulin treatment markedly stimulated the activity of a novel 31-kDa S6 kinase and the previously described 90-kDa ribosomal S6 kinase encoded by one of the rsk genes (p90rsk) in the 2-month control rats, while the effect was substantially reduced in the diabetic rats. In the 6-month diabetic rats, the basal phosphotransferase activities of both MAP kinases were depressed threefold or greater. This correlated with reductions in the amount of immunoreactive p42mapk and p44erk1 proteins in extracts from the diabetic rats. The basal activity of the 31-kDa S6 kinase activity was also reduced fourfold in the 6-month diabetic rats. Treatment of the 2-month diabetic rats with vanadyl sulfate resulted in euglycemia, prevented the increase in the basal activity of S6 kinase, and improved the activation of S6 kinase by insulin. Treatment of the 6-month diabetic rats with an organic vanadium compound, bis(maltolato)oxovanadium(IV), also produced euglycemia and a restoration of the basal S6 kinase activities in these rats. The present findings indicate that insulin resistance associated with long-term diabetes may be linked with depressed signaling through these kinases and that this can be rectified by vanadium-containing compounds.
Lasp-1 has been identified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in pancreas, intestine and gastric mucosa and is selectively expressed in cells within epithelial tissues. In the gastric parietal cell, cAMP-dependent phosphorylation induces the partial translocation of lasp-1 to the apically directed F-actin-rich canalicular membrane, which is the site of active HCl secretion. Lasp-1 is an unusual modular protein that contains an N-terminal LIM domain, a C-terminal SH3 domain and two internal nebulin repeats. Domain-based analyses have recently categorized this protein as an epithelial representative of the nebulin family, which also includes the actin binding, muscle-specific proteins, nebulin, nebulette and N-RAP. In this study, we show that lasp-1 binds to non-muscle filamentous (F) actin in vitro in a phosphorylation-dependent manner. In addition, we provide evidence that lasp-1 is concentrated within focal complexes as well as in the leading edges of lamellipodia and the tips of filopodia in non-transformed gastric fibroblasts. In actin pull-down assays, the apparent K(d) of bacterially expressed his-tagged lasp-1 binding to F-actin was 2 micro M with a saturation stoichiometry of approximately 1:7. Phosphorylation of recombinant lasp-1 with recombinant PKA increased the K(d) and decreased the B(max) for lasp-1 binding to F-actin. Microsequencing and site-directed mutagenesis localized the major in vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to S(99) and S(146). BLAST searches confirmed that both sites are conserved in human and chicken homologues. Transfection of lasp-1 cDNA encoding for alanine substitutions at S(99) and S(146), into parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1 to the intracellular canalicular region. In gastric fibroblasts, exposure to the protein kinase C activator, PMA, was correlated with the translocation of lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC, these data suggest that other mechanisms in addition to cAMP-dependent phosphorylation can mediate the translocation of lasp-1 to regions of dynamic actin turnover. The localization of lasp-1 to these subcellular regions under a range of experimental conditions and the phosphorylation-dependent regulation of this protein in F-actin rich epithelial cells suggests an integral and possibly cell-specific role in modulating cytoskeletal/membrane-based cellular activities.
We have previously found that C3H/He mice are a strain of mice that is resistant to endothelial dysfunction produced by Angiotensin II (Ang II). Interestingly, C3H/He mice carry a point mutation in the Toll‐Like Receptor 4 (TLR4) gene that results in defective TLR4‐mediated signaling. The goal of this study was to further explore the potential role of TLR4 in the hypertension and endothelial dysfunction produced by Ang II. C57Bl/6 (TLR4+/+ strain) and C57Bl/10ScN mice (a strain that carries a null‐mutation for the TLR4 gene) were infused with either vehicle or Ang II (1000 ng/kg/min) for 14 days. Baseline blood pressure was similar in C57Bl/6 and C57Bl/10ScN and on Day 14 of vehicle‐infusion. Ang II‐infusion produced hypertension in both C57Bl/6 and C57Bl/10ScN mice, however the degree of hypertension was significantly greater in C57Bl/6 than C57Bl/10ScN mice (164±6 vs. 147±9 mmHg, respectively). Acetylcholine produced concentration‐dependent relaxation that was similar in carotid arteries from vehicle‐infused C57Bl/6 and C57Bl/10ScN mice. In contrast, responses to acetylcholine were markedly impaired in Ang II‐infused C57Bl/6 mice. Responses to acetylcholine in Ang II‐infused C57Bl/10ScN mice were similar to their vehicle‐infused counterparts. Taken together, these findings demonstrate that TLR4 expression contributes to the hypertension and endothelial dysfunction produced by Ang II.
Hypertension is prevalent in 46% of the US adult population with 40% of cases being attributable to obesity. Obese individuals have high serum levels of very low-density lipoprotein (VLDL) and sphingosine-1-phosphate (S1P). VLDL and S1P have been shown to stimulate aldosterone production in multiple zona glomerulosa cell models, with aldosterone thought to be the link between hypertension and obesity. S1P is transported in blood bound to lipoproteins such as VLDL, low-density lipoprotein (LDL), and high-density lipoprotein (HDL); the VLDL particle contains the highest S1P levels. S1P in HDL has been shown to promote interactions between the scavenger receptor class B, type I (SR-BI) and S1P receptor 1 (S1PR1). We hypothesized that like HDL, VLDL will signal through S1PRs, upon binding to SR-BI; therefore, VLDL-induced aldosterone secretion will be inhibited by S1PR and SR-BI antagonists. Human adrenocortical (HAC15) cells were treated with VLDL and/or an S1PR1 antagonist (Ex26) or an anti-SR-BI blocking antibody for 24 h. Steroidogenic gene expression and aldosterone secretion were monitored by qRT-PCR and radioimmunoassay, respectively. Ex26 significantly inhibited VLDL-induced increases in CYP11B2 (22-fold) and StAR (1.5-fold) expression and aldosterone secretion (5-fold) by 43%, 10%, and 36%, respectively. Unexpectedly, the anti-SR-BI antibody enhanced VLDL-induced increases in CYP11B2 (29-fold) and NR4A1 (2.2-fold) expression by 17% and 33%, respectively; no effect was observed on VLDL-induced increases in StAR and NR4A2 expression. However, when the VLDL receptor was blocked with recombinant human LDL receptor related protein associated protein 1, VLDL-induced increases in CYP11B2 (2.8-fold) and NR4A2 (5.3-fold) expression were significantly reduced by 42% and 67%, respectively. Our results, therefore, indicate that VLDL signals through S1PR1, upon binding to the VLDL receptor and not SR-BI, to induce aldosterone synthesis and secretion. Our study warrants further investigation into VLDL-induced steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets like S1PR1 to potentially treat obesity-associated hypertension.
Tumor protein D52 is overexpressed in several cancers, including breast and prostate. In the GI tract, D52 (CSPP28, CRHSP28) is highly expressed in cells that undergo classical exocytosis, as pancreatic acinar, gastric chief and intestinal goblet cells, and appears to play a regulatory role in this process. Cholinergic stimulation or elevation of [Ca2+]i, but not PKC activation, lead to D52 phosphorylation on serine residues. The goal of this study was to identify the in vivo phosphorylation site(s) in D52 as well as the protein kinase(s) involved. T84 colon cancer cells expressing HA-tagged D52 were used as a model. To identify calcium-dependent phosphorylation site(s), cells were incubated with calcium ionophore and HA-tagged D52 was immunoprecipitated with anti-HA antibody. Ser136, a casein kinase 2 (CK2) consensus site, was identified by mass spectrometry as the calcium-dependent phosphorylation site and confirmed in 32P metabolic labeling experiments with ser to ala136 mutation. Both CK2 and D52 were immunolocalized to secretory vesicles. However, although carbachol stimulation of T84 cells induced protein kinase activation, as assessed by increased protein phosphorylation in cell extracts, CK2 activity per se was not increased. We hypothesize that a calcium-dependent alteration in protein-protein interactions provides a link for CK2 phosphorylation of D52. (Supported by NIH R01 DK31900).
In the GI tract, tumor protein D52 is highly expressed in cells that undergo classical exocytosis and has been implicated as a regulator of this process. We have shown that the cholinergic agonist, carbachol, and the calcium ionophore, ionomycin, increase the phosphorylation of S136 in the D52 protein in gastric mucosal cells. The goal of this study was to identify the protein kinase(s) involved. A phosphospecific antibody was produced and used to identify kinase(s) that specifically phosphorylate S136. D52 kinase activity in extracts from mouse and rabbit gastric glands and HEK293 cells was resolved on ion exchange and gel filtration columns. A single protein kinase with an apparent Mr of 46 kDa was identified using “in gel” assays with D52 as a substrate. The 46 kDa protein kinase bound to calmodulin Sepharose 4B, cross‐reacted with several pan‐specific CAMKII antibodies and, upon activation with Ca 2+ /calmodulin, also cross‐reacted with anti‐active CAMKII (pT 286/287 ) antibody. Carbachol‐stimulated phosphorylation of S136 was inhibited by the CAMKII inhibitor, KN93, with an IC50 of ∼20 μM and by the calmodulin antagonist, W7, with an IC50 of ∼30 μM. We conclude that a novel, low Mr CAMKII isoform is expressed in gastric mucosa and in kidney and that this protein kinase regulates calcium‐dependent D52 function(s) in these tissues. (Supported by NIH R01 DK31900).
We examined the distribution of mitogen-activated protein (MAP) kinase, S6 kinase, and casein kinase II (CK-II) in the muscle, spleen, brain, and testes of Wistar rats. It was observed that spleen extracts contained the highest activity of all the kinases. Anion-exchange chromatography of spleen extracts by a MonoQ column resolved a single peak of myelin basic protein phosphotransferase activity that eluted after the usual position of the previously described p42 and p44 MAP kinases. Immunoblotting of the peak fractions with anti-MAP kinase antibody did not detect any immunoreactive bands that coincided with the activity peak, suggesting that the activity may represent a potentially novel MAP kinase. The MonoQ fractionation also resolved a single peak of phosvitin phosphotransferase activity which coincided with the intensity of two immunoreactive bands of 39 and 43 kilodaltons that were detected with antibodies against CK-II. The chromatographic behaviour and immunoblotting data indicate that the phosvitin kinase peak represented CK-II and suggested that the rat spleen CK-II had a molecular structure of alpha alpha ' beta 2. Furthermore, using an intact rat model, we showed that the potentially novel spleen MAP kinase and CK-II were markedly activated following intravenous injection of insulin. The significance of these findings remains to be determined.
The goal of this study was to examine the role of interleukin‐6 (IL‐6), a pro‐inflammatory cytokine, in the endothelial dysfunction associated with aging in the cerebral circulation. Vascular responses were examined in basilar arteries from young (6 mo) and old (24 mo of age) wild‐type (WT) and IL‐6‐deficient mice. Dilatation to acetylcholine (ACh), an endothelium‐dependent agonist, was similar (P>0.05) in basilar arteries from young WT and young IL‐6 deficient mice. For example, 100 μM ACh produced 65±3% and 71±4% dilatation in basilar arteries from young WT and young IL‐6 deficient mice, respectively. With age, responses to ACh were impaired by ~100% in old WT mice as compared to their young counterparts. We have shown previously that this impairment of endothelial function in the basilar artery with age is mediated by reactive oxygen species (ROS). In contrast, the response to 100 μM ACh was partially reduced in old IL‐6 deficient mice, however the degree of impairment was significantly less than that in old WT mice (e.g., 100 μM ACh produced 38±4% and 0±0% dilatation in old IL‐6 deficient and old WT mice, respectively). Responses to nitroprusside (an endothelium‐independent agonist) were not affected (P>0.05) by genotype or aging. These findings suggest that IL‐6, in part and/or in conjunction with ROS, contributes to the endothelial dysfunction observed with aging in the cerebral circulation.
IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.
