The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.     

Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats

Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH.     

EP3, Prostaglandin E2 Receptor Subtype 3, Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage

EP3 is prostaglandin E2 receptor subtype 3 and mediates the activation of several signaling pathways, changing in cAMP levels, calcium mobilization, and activation of phospholipase C. Previous studies demonstrated a direct role for EP3 in various neurodegenerative disorders, such as stroke and Alzheimer disease. However, the distribution and function of EP3 in ICH diseases remain unknown. Here, we demonstrate that EP3 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). From the results of Western blot and immunohistochemistry, we obtained a significant up-regulation of EP3 in neurons adjacent to the hematoma following ICH. Up-regulation of EP3 was found to be accompanied by the increased expression of active caspase-3 and pro-apoptotic Bcl-2-associated X protein (Bax) and decreased expression of anti-apoptotic protein B cell lymphoma-2 (Bcl-2) in vivo and vitro studies. Furthermore, the expression of these three proteins reduced active caspase-3 and Bax expression, while increased Bcl-2 were changed after knocking down EP3 by RNA interference in PC12 cells, further confirmed that EP3 might exert its pro-apoptotic function on neuronal apoptosis. Thus, EP3 may play a role in promoting the neuronal apoptosis following ICH.     

Effects of RNA Interference of Atg4B on the Limited Proteolysis of LC3 in PC12 Cells and Expression of Atg4B in Various Rat Tissues

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, but the expression levels were not identical, but were dependent on the tissue, with the expression was high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.     

Effects of RNA interference of Atg4B on the limited proteolysis of LC3 in PC12 cells and expression of Atg4B in various rat tissues

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.     

Nemo-like Kinase (NLK) Involves in Neuronal Apoptosis after Traumatic Brain Injury

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact: and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase involved in neuronal apoptosis following TBI. In the model of TBI, western blot analysis, double immunofluorescent staining and immunohistochemistry were used to analyze the role of NLK in the process. The results showed a significant down-regulation of NLK and a concomitant up-regulation of caspase-3 during the early stage of TBI. In the model of glutamate inducing PC12 apoptosis, we analyzed the effect of over-expression of NLK on the neuronal cell line PC12 apoptosis by cck-8, western blot and TUNEL assays. Together with previous reports. We hypothesize NLK was related to the down-regulation of caspase-3 expression after TBI, and such an event may be associated with neuronal apoptosis.     

Nerve Growth Factor Determines Survival and Death of PC12 Cells by Regulation of the bcl-x, bax, and caspase-3 Genes

We investigated the effects of nerve growth factor (NGF) and NGF withdrawal on expression of members of the bcl-2 family of genes and caspase-3 in PC12 cells. NGF regulated several members of the bcl-2 family and caspase-3 in a manner consistent with its effect on apoptosis in PC12 cells. Levels of bcl-xl, bcl-xs, and caspase-3 mRNAs were increased by NGF treatment. The increases in caspase-3 and bcl-xs levels should have disposed the cells toward apoptosis but were opposed by the simultaneous increase in bcl-xl level. NGF withdrawal resulted in abrupt down-regulation of bcl-xl and up-regulation of bax, favoring apoptosis. Forced expression of bcl-xl after NGF withdrawal was sufficient to prevent cell death. Cell death was rapid when NGF was withdrawn after 5 days of treatment but relatively slow when NGF was withdrawn after only 1 or 2 days of treatment. This was consistent with the reduced accumulation of caspase-3 mRNA with shorter NGF treatments. These results indicate that Bcl-xl, Bcl-xs, Bax, and caspase-3 are important regulators of apoptosis in PC12 cells. Furthermore, regulation of their mRNA levels is implicated in the signal transduction of NGF.     

TLR4 signaling promotes the expression of VEGF and TGFbeta1 in human prostate epithelial PC3 cells induced by lipopolysaccharide

Chronic inflammation promotes tumor development and progression, and Toll-like receptors (TLRs) may play an important role in this process. In this study, we found that human prostate epithelial PC3 cells constitutively express TLR4 in mRNA and protein level. lipopolysaccharide (LPS) promotes the expression and secretion of immunosuppressive cytokine TGFβ₁ and proangiogenic factor VEGF in human prostate epithelial PC3 cells. We further elucidated that functionally activation of TLR4 is essential for the increased VEGF and TGFβ₁ mRNA expression in the cells. In addition, after LPS stimulation, the increased expression of NF-_KB p65 protein was also detected in human PC3 cells. Our results demonstrate that TLR4 expressed on human PC3 cells is functionally active, and may play important roles in promoting prostate cancer immune escape, survival, progression, and metastasis by inducing immunosuppressive and proangiogenic cytokines.     

Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding

The budding reactions of a number of enveloped viruses use the cellular machinery involved in the formation of the luminal vesicles of endosomal multivesicular bodies (MVB). Budding of these viruses is dependent on the presence of specific late-domain motifs in membrane-associated viral proteins. Such budding reactions usually involve ubiquitin and are blocked by expression of an ATPase-deficient form of VPS4, a cellular AAA+ ATPase believed to be required late in the MVB pathway for the disassembly/release of the MVB machinery. Here we examined the role of the MVB pathway in the budding of the late-domain-containing rhabdovirus vesicular stomatitis virus (VSV) and the alphavirus Semliki Forest virus (SFV). We tested early and late steps in the MVB pathway by depleting ubiquitin with the proteasome inhibitor MG-132 and by using cell lines inducibly expressing VPS4A or VPS4B protein. As previously shown, VSV budding was strongly dependent on ubiquitin. In contrast to the findings of previous studies with VPS4A, expression of ATPase-deficient mutants of either VPS4A or VPS4B inhibited VSV budding. Inhibition by VPS4 required the presence of the PPPY late domain on the VSV matrix protein and resulted in the accumulation of nonreleased VSV particles at the plasma membrane. In contrast, SFV budding was independent of both ubiquitin and the activity of VPS4, perhaps reflecting the important role of the highly organized envelope protein lattice during alphavirus budding.     

Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.     

RBM5 and p53 expression after rat spinal cord injury: Implications for neuronal apoptosis

RBM5 (RNA-binding motif protein 5), a nuclear RNA binding protein, is known to trigger apoptosis and induce cell cycle arrest by regulating the activity of the tumor suppressor protein p53. However, its expression and function in spinal cord injury (SCI) are still unknown. To investigate whether RBM5 is involved in central nervous system injury and repair, we performed an acute SCI model in adult rats in this study. Our results showed RBM5 was unregulated significantly after SCI, which was accompanied with an increase in the levels of apoptotic proteins such as p53, Bax, and active caspase-3. Immunofluorescent labeling also showed that traumatic SCI induced RBM5 location changes and co-localization with active caspase-3 in neurons. To further probe the role of RBM5, a neuronal cell line PC12 was employed to establish an apoptotic model. Knockdown of RBM5 apparently decreased the level of p53 as well as active caspase-3, demonstrating its pro-apoptotic role in neurons by regulating expressions of p53 and caspase-3. Taken together, our findings indicate that RBM5 promotes neuronal apoptosis through modulating p53 signaling pathway following SCI.     

Identification of an AAA ATPase VPS4B-dependent pathway that modulates epidermal growth factor receptor abundance and signaling during hypoxia

VPS4B, an AAA ATPase (ATPase associated with various cellular activities), participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (assessed with anchorage-independent three-dimensional multicellular spheroid culture) hypoxia induces VPS4B downregulation by the ubiquitin-proteasome system. Inhibition of VPS4B function by short hairpin VPS4B (sh-VPS4B) or expression of dominant negative VPS4B(E235Q) promotes anchorage-independent breast cancer cell growth and resistance to gefitinib, U0126, and genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied by increased EGFR accumulation and altered intracellular compartmentalization, is observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1 promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronic-hypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a nongenomic mechanism for EGFR overproduction in human breast cancer.     

X-linked mental retardation gene CUL4B targets ubiquitylation of H3K4 methyltransferase component WDR5 and regulates neuronal gene expression

CUL4B, encoding a scaffold protein for the assembly of Cullin4B-Ring ubiquitin ligase (CRL4B) complexes, is frequently mutated in X-linked mental retardation (XLMR) patients. Here, we show that CUL4B, but not its paralog, CUL4A, targets WDR5, a core subunit of histone H3 lysine 4 (H3K4) methyltransferase complexes, for ubiquitylation and degradation in the nucleus. Knocking down CUL4B increases WDR5 and trimethylated H3K4 (H3K4me3) on the neuronal gene promoters and induces their expression. Furthermore, CUL4B depletion suppresses neurite outgrowth of PC12 neuroendocrine cells, which can be rescued by codepletion of WDR5. XLMR-linked mutations destabilize CUL4B and impair its ability to support neurite outgrowth of PC12 cells. Our results identify WDR5 as a critical substrate of CUL4B in regulating neuronal gene expression and suggest epigenetic change as a common pathogenic mechanism for CUL4B-associated XLMR.     

Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells

Neuronal cell death after traumatic brain injury, Alzheimer’s disease and ischemic stroke may in part be mediated through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78 and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145 levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120 was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7 substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site D³⁴¹ detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2 and caspase-3/7.