Insulin regulates alternative splicing of PKCβII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., ...Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648–22654). Transient transfection of constitutively active Akt2 kinase promotes PKCβII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser86 on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCβII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCβII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCβI and -βII mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCβII levels in Akt(-/-), whereas PKCβI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCβII splicing minigene revealed defective βII exon inclusion. Co-transfection of the mutated SRp40 attenuated βII exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.
We recently reported on the enhanced tubular expression of two discrete isoforms of the MMP-2 (full length and N-terminal truncated, FL-MMP-2, NTT-MMP-2) in a murine model and human diabetic kidneys. ...In the present study, we examined in more detail the temporal and spatial distributions of MMP-2 isoform expression in murine models of Type 1 and Type 2 diabetes mellitus.
Diabetic models were streptozotocin (STZ)-induced diabetes (Type 1 diabetes mellitus) and db/db mice (Type 2 diabetes mellitus). We quantified the abundance of two isoforms of MMP-2 transcripts by qPCR. A spatial distribution of two isoforms of MMP-2 was analyzed semi-quantitatively according to time after injection of STZ and with increasing age of db/db mice. Furthermore, immunohistochemistry for nitrotyrosine was performed to examine a potential association between oxidative stress and MMP-2 isoform expression.
Both isoforms of MMP-2 were upregulated in whole kidneys from STZ and db/db mice. In the case of FL-MMP-2, mRNA levels significantly increased at 12 and 24 weeks in STZ mice, while the isoform expression was significantly increased only at 16 weeks, in the db/db mice. FL-MMP-2 protein levels increased in the cortices and outer medullae of both STZ and db/db mice as a function of the duration of diabetes. For NTT-MMP-2, mRNA levels increased earlier at 4 weeks in STZ mice and at 10 weeks of age in db/db mice. The expression of NTT-MMP-2 also increased, primarily in the cortices of STZ and db/db mice, as a function of the duration of diabetes. Quantitatively, these findings were consistent with the qPCR results in the case of NTT-MMP-2, respectively (STZ 24 weeks, 3.24 ± 3.70 fold; 16 weeks db/db, 4.49 ± 0.55 fold). In addition, nitrotyrosine was expressed primarily in cortex as compared to medulla as a function of the duration of diabetes similar to NTT-MMP-2 expression.
Two isoforms of MMP-2 are highly inducible in two diabetic murine models and become more abundant as a function of time. As the expression patterns were not the same in the two isoforms of MMP-2, it is possible that each isoform has a discrete role in the development of diabetic renal injury.
Uncaria sinensis (US) has been used in traditional Korean medicine to treat vascular disease and to relieve various neurological symptoms.
Scientific evidence related to the effectiveness or action ...mechanism of US on cerebrovascular disease has not been examined experimentally. Here, we investigated the cerebrovascular protective effect of US extracts on photothrombotic ischemic injury in mice.
US hexane extracts (HEUS), ethyl acetate extracts (EAEUS) and methanol extracts (MEUS) were administered intraperitoneally 30min before ischemic insults. Focal cerebral ischemia was induced in C57BL/6J mice and endothelial nitric oxide synthase knockout (eNOS KO) mice by photothrombotic cortical occlusion. We evaluated the infarct volume, neurological score and the activation of Akt and eNOS in ischemic brain.
HEUS more significantly reduced infarct volume and edema than did EAEUS and MEUS following photothrombotic cortical occlusion. HEUS produced decreased infarct volume and edema size, and improved neurological function in a concentration-dependent manner (10, 50, and 100mg/kg). However, HEUS did not reduce brain infarction in eNOS KO mice, suggesting that the protective effect of HEUS is primarily endothelium-dependent. Furthermore, HEUS (10–300μg/ml) produced a concentration-dependent relaxation in mouse aorta and rat basilar artery, which was not seen in eNOS KO mouse aorta, suggesting that HEUS cause vasodilation via an eNOS-dependent mechanism. This correlated with increased phosphorylation of Akt and eNOS in the brains of HEUS-treated mice.
HEUS prevent cerebral ischemic damage by regulating Akt/eNOS signaling. US, herbal medicine, may be the basis of a novel strategy for the therapy of stroke.
4-Hydroxynonenal (HNE), an end-product of membrane lipid peroxidation, has been suggested to mediate a number of oxidative stress-linked pathological events such as cellular apoptosis. However, ...little is known about the signals by which HNE induces vascular smooth muscle cell (VSMC) apoptosis. To elucidate the mechanism(s) involved in HNE-induced VSMC apoptosis, we investigated the importance of mitochondria as a potential source for reactive oxygen species (ROS). Exposure of VSMC to HNE (1–30
μM) showed an augmented apoptotic changes in a concentration-dependent manner in association with an increased production of ROS, both of which were significantly attenuated by mitochondrial inhibitors such as rotenone (0.1
μM) and stigmatellin (0.1
μM), but not affected by other oxidase inhibitors involving NADPH oxidase, xanthine oxidase and cyclooxygenase. In connection with these results, HNE-induced ROS generation was not observed in mitochondrial function-deficient (rho 0) VSMC. Taken together, these results suggest that mitochondrial dysfunction plays a key role in mediating HNE-induced VSMC apoptosis through an increased mitochondrial production of ROS.
Maladaptive repair after acute kidney injury (AKI) can predispose patients to chronic kidney disease (CKD). However, the molecular mechanism underlying the AKI-to-CKD transition remains unclear. The ...Akt signaling pathway has been reported to be involved in the pathological processes of both AKI and CKD. In this study, we investigated the role of Akt1 in a murine model of the AKI-to-CKD transition. Wild-type (WT) and Akt1−/− mice were subjected to unilateral ischemia-reperfusion injury (UIRI), with their kidneys harvested after two days and two, four, and six weeks after UIRI. The dynamic changes in tubulointerstitial fibrosis, markers of tubular epithelial-mesenchymal transition (EMT), and tubular apoptosis were investigated. Akt1 of the three Akt isoforms was activated during the AKI-to-CKD transition. After UIRI, tubulointerstitial fibrosis and tubular EMT were significantly increased in WT mice, but were attenuated in Akt1−/− mice. The expression of the transforming growth factor (TGF)-β1/Smad was increased in both WT and Akt1−/− mice, but was not different between the two groups. The levels of phosphorylated glycogen synthase kinase (GSK)-3β, Snail, and β-catenin in the Akt1−/− mice were lower than those in the WT mice. The number of apoptotic tubular cells and the expression of cleaved caspase-3/Bax were both lower in Akt1−/− mice than in WT mice. Genetic deletion of Akt1 was associated with attenuation of tubulointerstitial fibrosis, tubular EMT, and tubular apoptosis during the AKI-to-CKD transition. These findings were associated with TGF-β1/Akt1/GSK-3β/(Snail and β-catenin) signaling independent of TGF-β1/Smad signaling. Thus, Akt1 signaling could serve as a potential therapeutic target for inhibiting the AKI-to-CKD transition.
•Akt1, but not Akt2 or Akt3, was activated during the AKI-to-CKD transition.•Akt1 deletion attenuates the tubular apoptosis and tubular EMT.•Tubular EMT were related to the Akt1/GSK-3β/(Snail and β-catenin) pathways.
Angiotensin II (AngII) is a crucial hormone that affects vasoconstriction and exerts hypertrophic effects on vascular smooth muscle cells. Here, we showed that phosphatidylinositol 3-kinase-dependent ...calcium mobilization plays pivotal roles in AngII-induced vascular constriction. Stimulation of rat aortic vascular smooth muscle cell (RASMC)-embedded collagen gel with AngII rapidly induced contraction. AngII-induced collagen gel contraction was blocked by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) whereas ERK inhibitor (PD98059) was not effective. AngII-induced collagen gel contraction was significantly blocked by extracellular calcium depletion by EGTA or by nifedipine which is an L-type calcium channel blocker. In addition, AngII-induced calcium mobilization was also blocked by nifedipine and EGTA, whereas intracellular calcium store-depletion by thapsigargin was not effective. Finally, pretreatment of rat aortic ring with LY294002 and nifedipine significantly reduced AngII-induced constriction. Given these results, we suggest that PI3K-dependent activation of L-type calcium channels might be involved in AngII-induced vascular constriction.
Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal ...modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 SH2B3), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk (-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk (-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk (-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway.
Schisandra chinensis (SC), a member of the Magnoliaceae family, has been used to improve the vascular health for postmenopausal women in Korea. In order to provide some scientific rationales for such ...effectiveness, this study investigated the vascular effects of gomisin A (GA) from SC. In the endothelium (ED)-intact rings of rat thoracic aorta, GA (1 x 10 (-6) to 3 x 10 (-4) M) caused a concentration-dependent relaxation which was markedly attenuated not only by removal of ED but also by pretreatment with N(G)-nitro- L-arginine (10 (-4) M) or 1 H-1,2,4oxadiazol4,3- aquinoxalin-1-one (3 x 10 (-5) M). Direct measurement of nitrite, a metabolite of nitric oxide (NO), confirmed that NO production in isolated aorta was increased by GA. In the ED-denuded specimens, the relaxation by GA was not abolished but reduced significantly. The relaxation by GA in ED-denuded aortic rings were clearly inhibited by calyculin A (3 x 10 (-8) M), an inhibitor of MLC phosphatase. Furthermore, the phenylephrine-enhanced phosphorylation ratio of MLC was significantly attenuated by GA. Based on these results, it is suggested that GA induced vascular relaxation by partially activating ED-dependent NO pathway, and partially dephosphorylation of MLC.
This study was undertaken to explore the effects of aging on the kidneys in mouse models of diabetes and chronic kidney disease (CKD), and to compare the expression of two isoforms of matrix ...metalloproteinase-2 (MMP-2)-secretory full-length MMP-2 and intracellular N-terminal truncated MMP-2 (NTT-MMP-2)-in these models.
Two experimental ICR mouse models were used: a streptozotocin (STZ)-induced type 1 diabetes mellitus model and a 5/6 nephrectomized (5/6Nx) CKD model. The abundance of each isoform of MMP-2 was determined by quantitative polymerase chain reaction (qPCR), and functional analyses were conducted. Moreover, the protein levels of the two MMP-2 isoforms were determined semi-quantitatively by immunohistochemical staining, and their association with tissue damage was assessed.
Both isoforms of MMP-2 were upregulated in the kidney tissues of STZ-induced diabetic mice and 5/6Nx mice, irrespective of age. Characteristically, NTT-MMP-2 protein expression was elevated in old control mice, in line with the qPCR results. NTT-MMP-2 expression was limited to the renal cortex, and to the tubulointerstitial area rather than the glomerular area. In terms of tissue damage, tubulointerstitial fibrosis was more severe in old 5/6Nx mice than in their young counterparts, whereas glomerulosclerosis was comparable in old and young 5/6Nx mice.
The intracellular isoform of MMP-2 was induced by ageing, irrespective of the presence of diabetes or CKD, and its induction may be related to tubulointerstitial fibrosis in chronic kidney disease.
Endothelial cell-dependent vascular relaxation plays an important role in the regulation of blood pressure. Here, we show that stimulation of vascular endothelial cells with platelet-derived growth ...factor (PDGF) results in vascular relaxation through Akt1-dependent activation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Stimulation of both human umbilical artery endothelial cells and abdominal aortic vessels with PDGF induced NO production. PDGF-dependent production of NO was completely abolished by inhibition of phosphatidylinositol 3-kinase with wortmannin (100 nM). Stimulation of aortic vessels with PDGF resulted in the activation of Akt phosphorylation and eNOS phosphorylation: however, eNOS phosphorylation and production of NO were abolished in aortic vessels of mice lacking Akt1. PDGF strongly induced vascular relaxation in the presence of endothelium, and inhibition of NO production by
N
-nitro-
l
-arginine-methyl ester completely blocked PDGF-dependent vascular relaxation. In addition, PDGF-dependent relaxation was completely abolished by inhibition of PI3K with wortmannin (100 nM). Furthermore, vessels from Akt1 heterozygotes showed normal relaxation after PDGF stimulation, whereas vessels from Akt1 knockout littermates did not respond to PDGF stimulation. Finally, administration of PDGF (5 ng/ml) significantly lowered blood pressure in Akt1 heterozygotes, whereas a blood pressure-lowering effect was not observed in Akt1 knockout littermates. These results suggest that Akt1 regulates blood pressure through regulation of vascular relaxation by eNOS phosphorylation and subsequent production of NO.
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