DHA Attenuates Hypoxia/Reoxygenation Injury by Activating SSeCKS in Human Cerebrovascular Pericytes.

Docosahexaenoic acid (DHA) can alleviate cerebral ischemia/reperfusion injury by reducing blood-brain barrier permeability and maintaining its integrity, accompanied by an increased Ang-1/Ang-2 ratio; however, the underlying mechanisms of these effects remain unclear. Src-suppressed C kinase substrates (SSeCKS), a substrate of protein kinase C, plays an important role in maintaining cell junctions and cell morphology and regulating cell permeability. However, whether DHA can increase SSeCKS expression and then mediate the Ang-1/Ang-2 ratio still needs to be studied. Human cerebrovascular pericytes (HBVPs) cultured in vitro were divided into groups, treated with or without DHA along with SSeCKS siRNA to knockdown SSeCKS expression, and then subjected to 24 h of hypoxia followed by 6 h of reoxygenation. Cell viability; lactate dehydrogenase (LDH) release; and Ang-1, Ang-2 and VEGF activity were detected by using ELISA kits. The apoptosis rate was assessed by TUNEL flow cytometry. Expression of the SSeCKS, Ang-1, Ang-2 and VEGF proteins was evaluated by western blotting. Pretreatment with 10 µM or 40 µM DHA efficiently attenuated hypoxia/reoxygenation (H/R) injury by activating SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, as evidenced by decreased LDH release and apoptotic rates and increased HBVPs viability. Meanwhile, after we used SSeCKS siRNA to knock down SSeCKS protein expression, the protective effect of DHA on HBVPs following H/R injury was reversed. In conclusion, DHA can activate SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, thus reducing H/R injury.

Effect of the SSeCKS-TRAF6 interaction on gastrodin-mediated protection against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced astrocyte activation and neuronal death.

The ubiquitous environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to trigger neurotoxicity. In this study, we investigated the protective effects of gastrodin on TCDD-induced neurotoxicity and the underlying molecular mechanisms. The results show that gastrodin decreased cell viability, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release, and inducible nitrix oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) expression in TCDD-treated C6 cells. TCDD stimulated NF-κB signalling activation, demonstrated by increased p-NF-κB expression and translocation of nuclear Factor kappa B (NF-κB) to the nucleus. TCDD did not affect TRAF6 protein expression but enhanced the attenuated the Src-suppressed-C Kinase Substrate (SSeCKS)-tumor necrosis factor receptor-associated factor 6 (TRAF6) interaction, thereby triggering NF-κB signalling activation. Gastrodin inhibited TCDD-induced NF-κB signalling activation by lessening the SSeCKS-TRAF6 interaction in vitro. Gastrodin attenuated SSeCKS-TRAF6 interaction in vivo and protected mice from NF-κB signalling activation following TCDD exposure. Finally, gastrodin blocked the apoptosis of PC12 neuronal cells induced by medium conditioned with TCDD-treated astrocytes. In summary, gastrodin inhibited TCDD-induced NF-κB signalling activation by lessening the SSeCKS-TRAF6 interaction, resulting in attenuated astrocyte activation and subsequent neuronal apoptosis. These findings will contribute to an improved understanding of TCDD-induced neurotoxicity and strategies to antagonise it using gastrodin.

SSeCKS/Gravin/AKAP12 Inhibits PKCζ-Mediated Reduction of ERK5 Transactivation to Prevent Endotoxin-Induced Vascular dysfunction.

SSeCKS/Gravin/AKAP12 is a protein kinase C (PKC) substrate that inhibits the activity of PKC through binding with it. SSeCKS is expressed in vascular endothelial cells (ECs). The atypical PKC isoform ζ (PKCζ) is a pathologic mediator of endothelial dysfunction. However, the functional significance of SSeCKS/PKCζ dimerization in the vascular endothelium remains poorly understood. Given this background, we investigated the effects of SSeCKS on endothelial dysfunction and elucidated the possible mechanism involved. Vascular endothelial dysfunction and inflammatory changes were induced by treatment with bacterial endotoxin lipopolysaccharide (LPS, a vascular endothelial toxicity inducer). LPS can increase the level of SSeCKS. However, we also found that depletion of SSeCKS aggravated the LPS-induced vascular endothelial dysfunction, upregulated pro-inflammatory proteins and phosphorylation level of PKCζ, increased ROS formation, decreased extracellular-signal-regulated kinase 5 (ERK5) transcriptional activity, and reduced eNOS expression. Further examination revealed that depletion of SSeCKS increased PKCζ/ERK5 dimerization. These findings provide preliminary evidence that the expression of SSeCKS induced by LPS, as a negative feedback mechanism, has the potential to improve endothelium-dependent relaxation in vascular disease conditions by inhibiting PKCζ-mediated reduction of ERK5 transactivation.

SSeCKS promoted lipopolysaccharide-sensitized astrocytes migration via increasing ß-1,4-galactosyltransferase-I activity.

Astrocytes migration is essential in the formation of the glial scar during the injury response process of the central nervous system (CNS) especially during inflammation. Integrin ß1 is part of the extracellular matrix receptors in the CNS and it has been reported that integrin ß-deficient astrocytes randomly migrate into wounds. Previous studies have found that ß-1,4 Galactosyltransferase-I (ß-1,4-GalT-I) enhanced the ß-1,4-galactosylation of integrin ß1. Src-suppressed C kinase substrate (SSeCKS) is an inflammatory response protein which functionally interacts with ß-1,4 Galactosyltransferase-I (ß-1,4-GalT-I). In this study we aim to investigate the role of SSeCKS and ß-1,4-GalT-I in the migration of astrocytes during lipopolysaccharide (LPS)-induced inflammation. Coimmunoprecipitation and immunofluorescence assays have demonstrated that SSeCKS and ß-1,4-GalT-I were significantly enhanced in LPS-treated astrocytes and their interactions may occur in the Trans-Golgi Network. Lectin blot showed that the knockdown of ß-1,4-GalT-I could inhibit the ß-1,4-galactosylation of glycoproteins including integrin ß1 with and without LPS, and that SSeCKS knockdown inhibits the ß-1,4-galactosylation of glycoproteins including integrin ß1 only in LPS-induced astrocytes. Additionally, wound healing assays indicated that ß-1,4-GalT-I knockdown could inhibit astrocytes migration with and without LPS but SSeCKS inhibited cell migration only when LPS was present. Therefore our findings suggest that SSeCKS affects astrocytes migration by regulating the ß-1,4-galactosylation of glycoproteins including integrin ß1, via ß-1,4-GalT-I expression in LPS-sensitized astrocytes.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[Braf V600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts.

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [1]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[BrafV600E] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naïve WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naïve KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-ß-galactosidase, p21waf1 and p16ink4a, and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.

SSeCKS/AKAP12 induces repulsion between human prostate cancer and microvessel endothelial cells through the activation of Semaphorin 3F.

Metastasis remains the primary cause of prostate cancer related death. Cancer cells need to contact endothelial cells and disrupt endothelial junctions to cross the endothelium for invasion and metastasis. The suppression of heterotypic repulsion between cancer and endothelial cells allows cancer cells to invade into the surrounding tissue. Here, we demonstrate that SSeCKS/AKAP12 induced repulsion between human prostate cancer and microvessel endothelial cells, which was mediated by an angiogenesis inhibitor Semaphorin 3F. Moreover, we examined AKAP12 and Semaphorin 3F mRNA expression in 42 prostate cancer and 30 benign prostatic hyperplasia tissue samples, and found that the expression of AKAP12 and Semaphorin 3F mRNA was inversely associated with the degree of aggressiveness of prostate cancer cells and tissues. An ordinal logistic regression analysis indicates that there is a positive association between the expression of AKAP12 and Semaphorin 3F in prostate cancer, suggesting that the activation of Semaphorin 3F by SSeCKS/AKAP12 may be involved in prostate cancer progression and metastasis.

Xijiao Dihuang decoction combined with Yinqiao powder inhibits TNF-α-induced permeability increase in PMVEC via PKC-SSeCKS pathway / 中国病理生理杂志

AIM:To investigate the molecular mechanism of Xijiao Dihuang decoction combined with Yinqiao powder (XDY) in treating viral pneumonia, and the effects of XDY on TNF-α-induced permeability in pulmonary microvascular endothelial cells (PMVEC) and the role of PKC-SSeCKS pathway involved.METHODS:The electric conductivity method was used to detect transendothelial electrical resistance (TER) of primarily cultured PMVEC on Transwell chamber at different time points to determine the permeability of PMVEC.After pretreatment for 24 h, the activity of PKC, TER, and the expression of SSeCKS at mRNA and protein levels were detected.Laser scanning confocal microscopy was used to observe the location of SSeCKS and construction of F-actin in PMVEC.RESULTS:The permeability of PMVECs induced by TNF-α reached the peak at 24 h.Compared with control group, the TER in TNF-α group was decreased, and the activity of PKC was increased.Compared with TNF-α group, the activity of PKC in TNF-α with PKC inhibitor group and TNF-α with XDY group was decreased, while the TER was increased, without difference from control group.Compared with control group, the mRNA expression of SSeCKS and phospho-SSeCKS was increased in PMVEC of TNF-α group, but decreased in TNF-α with XDY group compared with TNF-α group.In control group, F-actin was mainly located around the nucleus and at cytoplasmic borders of PMVEC, forming the dense peripheral bundle, and SSeCKS was evenly scattered in the cell.In TNF-α group, the dense peripheral bundle of F-actin surrounding the cells almost disappeared, and SSeCKS was concentrated around the nucleus.Compared with TNF-α group, the distribution and the structure of F-actin and SSeCKS nearly returned to normal in TNF-α with XDY group.CONCLUSION:XDY inhibits the activation of PKC signaling pathway in PMVEC caused by TNF-α to reduce the mRNA expression of SSeCKS and the phosphorylation of SSeCKS, thus preventing the deformation of endothelial cells and reducing the permeability of PMVEC.

Vascular endothelial growth factor: an attractive target in the treatment of hypoxic/ischemic brain injury.

Cerebral hypoxia or ischemia results in cell death and cerebral edema, as well as other cellular reactions such as angiogenesis and the reestablishment of functional microvasculature to promote recovery from brain injury. Vascular endothelial growth factor is expressed in the central nervous system after hypoxic/ischemic brain injury, and is involved in the process of brain repair via the regulation of angiogenesis, neurogenesis, neurite outgrowth, and cerebral edema, which all require vascular endothelial growth factor signaling. In this review, we focus on the role of the vascular endothelial growth factor signaling pathway in the response to hypoxic/ischemic brain injury, and discuss potential therapeutic interventions.

HSPA12B regulates SSeCKS-mediated astrocyte inflammatory activation in neuroinflammation.

Reactive astrocytosis has been considered either beneficial or detrimental effection in neuroinflammatory disease. HSPA12B, a new member belongs to the 70-kDa family of heat shock proteins (HSP70) which could modulate inflammatory response, also shows an connection with the astrocyte activation. Recently, it was reported that Src-Suppressed-C Kinase Substrate (SSeCKS) was detected in heat shock protein A12B (HSPA12B) interacting proteins using a yeast 2-hybrid system. SSeCKS, a major Lipopolysaccharide (LPS) response protein, has been involved in regulating astrocyte activation via production of proinflammatory factor in CNS inflammation. In this study, we found HSPA12B might regulate the expression and activity of SSeCKS to promote astrocyte inflammatory activation and release of inflammatory mediators, such as TNF-α and IL-1ß in spinal cord primary astroglial cultures exposed to LPS treatment. The promoting mechanism of interaction between HSPA12B and SSeCKS on LPS-induced astrocyte activation was mediated via the activation of JNK and p38 signaling pathways but not ERK1/2 MAPK signaling pathway. HSPA12B binded to SSeCKS via its both N terminus consisted of amino acids 1-330 and C terminus consisted of amino acids 1278-1596. And, in vivo, we confirmed the interaction between HSPA12B and SSeCKS of astrocyte activation in the pathogenesis of EAE. The regulatory mechanisms of HSPA12B-SSeCKS interaction may possibly be the key therapeutic strategy of neuroinflammatory disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes astrocyte activation and the secretion of tumor necrosis factor-α via PKC/SSeCKS-dependent mechanisms.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro-inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src-Suppressed-C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)-mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC-induced SSeCKS activation in TCDD-triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD-induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho-p65 expression, and tumor necrosis factor-α secretion in astrocytes. In addition, pre-treatment with PKC inhibitor also attenuated TCDD-induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD-triggered tumor necrosis factor-α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS-dependent mechanisms, highlighting that astrocytes might be important target of TCDD-induced neurotoxicity. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits neurotoxic effects. Here, we show TCDD induces pro-inflammatory responses in astrocytes. TCDD initiates an increase of [Ca2+]i, followed by the activation of PKC, which then induces the activation of Src-suppressed C-kinase substrate (SSeCKS). SSeCKS promotes NF-κB activation and the secretion of TNF-α and nitric oxide in astrocytes.

Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin.

A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC.

Emerging Roles for SSeCKS/Gravin/AKAP12 in the Control of Cell Proliferation, Cancer Malignancy, and Barriergenesis.

Emerging data suggest that SSeCKS/Gravin/AKAP12 ("AKAP12"), originally identified as an autoantigen in cases of myasthenia gravis, controls multiple biological processes through its ability to scaffold key signaling proteins such as protein kinase (PK) C and A, calmodulin, cyclins, phosphoinositides, "long" ß-1,4 galactosyltransferase (GalTase) isoform, Src, as well as the actin cytoskeleton in a spatiotemporal manner. Specialized functions attributed to AKAP12 include the suppression of cancer malignancy, especially aspects of metastatic progression, regulation of blood-brain and blood-retina barrier formation, and resensitization of ß2-adrenergic pain receptors. Recent data identify a direct role for AKAP12 in cytokinesis completion, further suggesting a function as a negative regulator of cell senescence. The current review will discuss the emerging knowledge base of AKAP12-related biological roles and how the factors that affect AKAP12 expression or that interact with AKAP12 at the protein level control cancer progression and blood-tissue barrier formation.

THE CHANGE OF SSeCKS AFTER THE TRANSECTION OF RAT SCIATIC NERVE / 解剖学报

Objective To investigate the change of SSeCKS in sciatic nerve after the transection of rat sciatic nerve and its significance. Methods The sciatic nerve transection model of Sprague-Dawley rat was made.We tested the change of SSeCKS by Western blotting and immunohistochemistry. Results It showed from Western blotting that after injury,SSeCKS in the proximal lesion site increased gradually and reached its peak at 2d,then it began to decrease,while in the distal lesion site,SSeCKS peaked at 12h and then declined.Immunohistochemistry showed SSeCKS mainly located in break site,in a cluster expression pattern,while in the uninjury region,the level of SSeCKS was significantly lower and the distribution was uniform.The result of double immunofluorescent staining showed that SSeCKS partly colocalized with S100,NF200 and GAP43.Conclusion The transection of rat sciatic nerve can cause the change of SSeCKS and it might be involved in transduction of some harm stimulate signal moleculars after the injury.It may be also related with the nerve regeneration and function repair.

EFFECTS OF LIPOPOLYSACCHARIDE ON THE EXPRESSION OF SRC-SUPPRESSED C KINASE SUBSTRATE IN CULTURED ASTROCYTES / 解剖学报

Objective To investigate the effects of lipopolysaccharide(LPS) on Src-suppressed C Kinase Substrate(SSeCKS) in cultured astrocytes. Methods Purified astrocytes were randomly divided into three groups:control group,singly stressed and multiple stressed groups.The expression of SSeCKS was detected by Real time RT-PCR and Western blotting,while immunocytochemistry was used to investigate the subcellular localization of it. Results Real time RT-PCR indicated that,after 12 hours incubation,both 100 ?g/L and 1mg/L LPS were able to elevate the levels of SSeCKS mRNA compared with control group,while 1mg/L LPS did not have a stronger effect than 100 ?g/L.Western blotting analysis showed 100 ?g/L LPS significantly increased the expression of SSeCKS.In time response experiments,the levels of SSeCKS expression enhanced three hours after the stimulation,peaked at the sixth hour,coincident with its rapid phosphorylation,and remained high until the 24th hour.Immunocytochemistry suggested a perinuclear translocation of SSeCKS,while the PKC inhibitor RO-31-8220 blocked it.Conclusion In cultured astrocytes,LPS can enhance the expression of SSeCKS,increase its phosphorylation level and change its subcellular localization.These effects are correlated with the PKC pathway,which indicates SSeCKS might participate in the signal transduction of inflammation in cultured astrocytes.