No abstract available

Inflammation is well established to significantly impact metabolic diseases. The inflammatory protease caspase-1 has been implicated in metabolic dysfunction; however, a potential role for the related inflammatory caspases is currently unknown. In this study, we investigated a role for caspase-11 and caspase-12 in obesity and insulin resistance. Loss of caspase-12 in two independently generated mouse strains predisposed mice to develop obesity, metabolic inflammation, and insulin resistance, whereas loss of caspase-11 had no effect. The use of bone marrow chimeras determined that deletion of caspase-12 in the radio-resistant compartment was responsible for this metabolic phenotype. The Nlrp3 inflammasome pathway mediated the metabolic syndrome of caspase-12-deficient mice as ablation of Nlrp3 reversed Casp12(-/-) mice obesity phenotype. Although the majority of people lack a functional caspase-12 because of a T(125) single nucleotide polymorphism that introduces a premature stop codon, a fraction of African descendents express full-length caspase-12. Expression of caspase-12 was linked to decreased systemic and adipose tissue inflammation in a cohort of African American obese children. However, analysis of the Dallas Heart Study African American cohort indicated that the coding T(125)C single nucleotide polymorphism was not associated with metabolic parameters in humans, suggesting that host-specific differences mediate the expressivity of metabolic disease.

Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-gamma but not by IFN-alpha or -beta. The effect is increased further when IFN-gamma is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand-, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress-inducing agent Tg whether they express caspase-12 or not.

Mycobacterium fortuitum is a natural fish pathogen. It induces apoptosis in headkidney macrophages (HKM) of catfish, Clarias sp though the mechanism remains largely unknown. We observed M. fortuitum triggers calcium (Ca2+) insult in the sub-cellular compartments which elicits pro-apototic ER-stress factor CHOP. Alleviating ER-stress inhibited CHOP and attenuated HKM apoptosis implicating ER-stress in the pathogenesis of M. fortuitum. ER-stress promoted calpain activation and silencing the protease inhibited caspase-12 activation. The study documents the primal role of calpain/caspase-12 axis on caspase-9 activation in M. fortuitum-pathogenesis. Mobilization of Ca2+ from ER to mitochondria led to increased mitochondrial Ca2+ (Ca2+)m load,, mitochondrial permeability transition (MPT) pore opening, altered mitochondrial membrane potential (ΔΨm) and cytochrome c release eventually activating the caspase-9/-3 cascade. Ultra-structural studies revealed close apposition of ER and mitochondria and pre-treatment with (Ca2+)m-uniporter (MUP) blocker ruthenium red, reduced Ca2+ overload suggesting (Ca2+)m fluxes are MUP-driven and the ER-mitochondria tethering orchestrates the process. This is the first report implicating role of sub-cellular Ca2+ in the pathogenesis of M. fortuitum. We summarize, the dynamics of Ca2+ in sub-cellular compartments incites ER-stress and mitochondrial dysfunction, leading to activation of pro-apoptotic calpain/caspase-12/caspase-9 axis in M. fortuitum-infected HKM.

Amyloid-beta (Abeta) neurotoxicity is believed to contribute to the pathogenesis of Alzheimer's disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Abeta neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)-resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Abeta increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2-mediated cell death. Finally, we find that E2-25K/Hip-2-deficient cortical neurons are resistant to Abeta toxicity and to the induction of ER stress and caspase-12 expression by Abeta. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress-mediated Abeta neurotoxicity.

Caspase-12 (CASP12) modulates the susceptibility to sepsis. In humans, the "C" allele at CASP12 rs497116 has been associated with an increased risk of sepsis. Instead, the derived "T" allele encodes for an inactive caspase-12. Interestingly, Eurasians are practically fixed for the inactive variant, whereas in Sub-Saharan Africa the active variant is still common (~24%). This marked structure has been explained as a function of the selective advantage that the inactive caspase-12 confers by increasing resistance to infection. As regards to both when positive selection started acting and as to the speed with which fixation was achieved in Eurasia, estimates depend on the method and assumptions used, and can vary substantially. Using experimental evidence, we propose that, least in Eurasia, the increase in the frequency of the T allele might be related to the selective pressure exerted by the increase in zoonotic diseases transmission caused by the interplay between increased human population densities and a closer contact with animals during the Neolithic. METHODOLOG/PRINCIPAL FINDINGS: We genotyped CASP12 rs497116 in prehistoric individuals from 6 archaeological sites from the North of the Iberian Peninsula that date from Late Upper Paleolithic to Late Neolithic. DNA extraction was done from teeth lacking cavities or breakages using standard anti-contamination procedures, including processing of the samples in a positive pressure, ancient DNA-only chamber, quantitation of DNAs by qPCR, duplication, replication, genotyping of associated animals, or cloning of PCR products. Out of 50, 24 prehistoric individuals could finally be genotyped for rs497116. Only the inactive form of CASP12 was found.

Genistein, a soy-derived isoflavone, has been suggested for breast cancer prevention; however, use of soy products for this purpose remains controversial. Genistein has been reported to regulate growth of tumor cells, although the involved molecular mechanisms are not defined. Here we report that genistein induces apoptosis in breast cancer cells via activation of the Ca2+ -dependent proapoptotic proteases, mu-calpain, and caspase-12. The treatment of MCF-7 breast cancer cells with genistein induced a sustained increase in concentration of intracellular Ca2+ resulting from depletion of the endoplasmic reticulum Ca2+ stores. This increase in Ca2+ was associated with activation of mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 substrates and antibodies to active (cleaved) forms of the enzymes. Selective inhibition of Ca2+ binding sites of mu-calpain, forced increase of the cytosolic Ca2+ buffering capacity, and caspase inhibition decreased apoptotic indices in the genistein-treated cells. Our results suggest that Ca2+ -dependent proteases are potential targets for genistein in breast cancer cells and that the cellular Ca2+ regulatory activity of genistein underlies its apoptotic mechanism.

Caspases are evolutionary conserved proteases traditionally known as participating in apoptosis and inflammation but recently discovered also in association with other processes such as proliferation or differentiation. This investigation focuses on caspase-12, ranked among inflammatory caspases but displaying other, not yet defined functions. A screening analysis pointed to statistically significant (P < 0.001) increase in expression of caspase-12 in a decisive period of mandibular bone formation when the original mesenchymal condensation turns into vascularized bone tissue. Immunofluorescence analysis confirmed the presence of caspase-12 protein in osteoblasts. Therefore, the osteoblastic cell line MC3T3-E1 was challenged to investigate any impact of caspase-12 on the osteogenic pathways. Pharmacological inhibition of caspase-12 in MC3T3-E1 cells caused a statistically significant decrease in expression of some major osteogenic genes, including those for alkaline phosphatase, osteocalcin and Phex. This downregulation was further confirmed by an alkaline phosphatase activity assay and by a siRNA inhibition approach. Altogether, this study demonstrates caspase-12 expression and points to its unknown physiological engagement in bone cells during the course of craniofacial development.

The unfolded protein response (UPR) is implicated in many neurodegenerative disorders including Alzheimer, Parkinson and prion diseases, and the leukodystrophy, Pelizaeus-Merzbacher disease (PMD). Critical features of degeneration in several of these diseases involve activation of cell death pathways in various neural cell populations, and the initiator caspase 12 has been proposed to play a central role. Accordingly, pharmacological strategies to inhibit caspase 12 activity have received remarkable attention in anticipation of effecting disease amelioration. Our investigation in animal models of PMD demonstrates that caspase 12 is activated following accumulation of mutant proteins in oligodendrocytes; however, eliminating caspase 12 activity does not alter pathophysiology with respect to levels of apoptosis, oligodendrocyte function, disease severity or life span. We conclude that caspase 12 activation by UPR signaling is an epiphenomenon that plays little discernable role in the loss of oligodendrocytes in vivo and may portend the inconsequence of caspase 12 to the pathophysiology of other protein conformational diseases.

The mechanisms underlying the potentially beneficial effect of bone marrow stem cells (BMSCs) on spinal cord injury (SCI) are unknown. Therefore, the aim of the present study was to explore the protective effect of BMSCs in rats with SCI. A total of 45 adult male Sprague-Dawley rats were randomly divided into three groups; the SCI group (n=15), the BMSC group (n=15) and the sham-operation group (n=15). In the SCI and BMSC treatment groups, a modified Allen's weight-drop technique was used to induce SCI. The BMSC treatment group received an injection of BMSCs using a microneedle into the epicenter of the spinal cord 24 h after injury. Rats in the sham-operation group were not subjected to SCI; however, the corresponding vertebral laminae were removed. Seven days after transplantation, a rapid recovery was observed in the Basso, Beattie and Bresnahan (BBB) scores of the BMSC treatment group, whereas the BBB scores in the SCI group remained low (P<0.05). Caspase-12 expression in the SCI group was increased compared with that in the sham-operation group, whereas caspase-12 expression was attenuated 24 h after transplantation in the BMSC treatment group (P<0.05). In conclusion, the transplantation of BMSCs may improve locomotor function and attenuate caspase-12 expression following SCI. Therefore, it is likely to be an effective strategy for preventing severe injury of the spinal cord.

Calpains and caspases are two cysteine protease families that play important roles in regulating pathological cell death. Here, we report that m-calpain may be responsible for cleaving procaspase-12, a caspase localized in the ER, to generate active caspase-12. In addition, calpain may be responsible for cleaving the loop region in Bcl-xL and, therefore, turning an antiapoptotic molecule into a proapoptotic molecule. We propose that disturbance to intracellular calcium storage as a result of ischemic injury or amyloid beta peptide cytotoxicity may induce apoptosis through calpain- mediated caspase-12 activation and Bcl-xL inactivation. These data suggest a novel apoptotic pathway involving calcium-mediated calpain activation and cross-talks between calpain and caspase families.

Stroke is one of the leading causes of death worldwide. Accumulating evidence suggests that NLRP3 inflammasome activation plays an important role in ischemic stroke injury. However, the existence of the NLRP3 inflammasome in astrocytes remains controversial. In this study, we demonstrated the presence of the NLRP3 inflammasome in primary mouse astrocytes and investigated the role of caspase-12 in NLRP3 inflammasome activation and cell injury in an in vitro astrocyte oxygen-glucose deprivation (OGD) model. Astrocytes exposed to 2, 3, and 4 h of OGD exhibited increased cell injury and apoptosis, and the protein levels of caspase-12, cleaved caspase-3, NLRP3 inflammasome components, and IL-1β were also significantly elevated. Interestingly, pretreatment with the caspase-12-specific inhibitor Z-ATAD-FMK attenuated cell injury and apoptosis and decreased the levels of NLRP3, caspase-1, IL-1β, and cleaved caspase-3 in the OGD group. In conclusion, Z-ATAD-FMK protected astrocytes against OGD-induced cell death and inhibited NLPR3-inflammasome activation. Our results indicate that caspase-12 and its potential regulation of NLRP3 inflammasome activation might be a promising target for treatment of ischemic stroke.

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.

The human caspase-12 gene is polymorphic for the presence or absence of a stop codon, which results in the occurrence of both active (ancestral) and inactive (derived) forms of the gene in the population. It has been shown elsewhere that carriers of the inactive gene are more resistant to severe sepsis. We have now investigated whether the inactive form has spread because of neutral drift or positive selection. We determined its distribution in a worldwide sample of 52 populations and resequenced the gene in 77 individuals from the HapMap Yoruba, Han Chinese, and European populations. There is strong evidence of positive selection from low diversity, skewed allele-frequency spectra, and the predominance of a single haplotype. We suggest that the inactive form of the gene arose in Africa approximately 100-500 thousand years ago (KYA) and was initially neutral or almost neutral but that positive selection beginning approximately 60-100 KYA drove it to near fixation. We further propose that its selective advantage was sepsis resistance in populations that experienced more infectious diseases as population sizes and densities increased.

Caspase-12 (Casp12), an inflammatory caspase, functions as a dominant-negative regulator of inflammatory responses and is associated with the signaling of apoptosis. However, the physiological function of Casp12 presented in cancer cells is still unclear. This study demonstrated that overexpression of Casp12 mediated IκBα degradation and significantly increased NF-κB activity. Exposure of human nasopharyngeal carcinoma (NPC) cells to phorbol-12-myristate-13-acetate (PMA) increased the levels of Casp12 and MMP-9 resulting in NPC cell invasion. Target suppression of Casp12 by small interfering RNA (siRNA) or an inhibitor of Casp12 markedly decreased the level of PMA-induced MMP-9 protein and cell invasion. Moreover, suppression of Casp12 significantly inhibited the basal activity of NF-κB and decreased the PMA-induced NF-κB reporter activity. The effect of Casp12 on NF-κB activation was indicated via the post-translational degradation of IκB. This study revealed that a critical role of Casp12 on the activation of NF-κB via IκBα degradation which provides a link between inflammatory and aggressive invasion in NPC cells.

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca(2+) signal, sustained increase in intracellular Ca(2+), triggers apoptotic cell death via activation of Ca(2+)-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is mediated through Ca(2+) signaling. Here, we report that 1,25(OH)(2)D(3) induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca(2+)-dependent calpain and Ca(2+)/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)(2)D(3) induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca(2+). The increase in Ca(2+) was associated with induction of apoptosis and activation of mu-calpain and caspase-12. Our results demonstrate that Ca(2+)-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)(2)D(3) in these cells are Ca(2+)-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.

As a potent novel free radical scavenger, edaravone has been reported to have neuroprotective effects in both animals and humans, although the underlying mechanisms remain unclear. In our study, we generated a culture of almost pure neurons, which were either left untreated or prophylactically treated with edaravone, then exposed to 50 μM glutamate for 10 min. Flow Cytometry analysis was performed to quantify the percentage of apoptotic cells. Ultrastructural changes in the endoplasmic reticulum were observed by electron microscopy. Immunofluorescence and western blotting for activation of selected related molecules, including PERK (pancreatic ER stress kinase, PERK), eIF2α (eukaryotic initiation factor 2 alpha, eIF2α), activating ATF4 (transcription factor 4, ATF4), and caspase-12 were examined. In Glutamate-treated group, the sequential activation of PERK, eIF2α, ATF4 and caspase-12 could be observed at 2h, and peaked at 24h. However, treatment with edaravone was able to prevent these changes. In addition, the morphology of the endoplasmic reticulum was better preserved and the percentage of apoptotic cells was lower in cells treated with edaravone. In summary, our results indicate that ISR (PERK/eIF2/ATF4 integrated stress response, ISR) plays an important role in glutamate-induced nerve cells death, and that edaravone could protect neurons against glutamate-induced endoplasmic reticulum stress.

Galanin (GAL) plays key role in many pathophysiological processes, but its role in ischemic stroke remains unclear. Here, the models of 1 h middle cerebral artery occlusion (MCAO)/1-7 d reperfusion (R)-induced ischemic stroke and in vitro cell ischemia of 1 h oxygen-glucose deprivation (OGD)/24 h reoxygenation in primary cultured cortical neurons were used to explore GAL's effects and its underlying mechanisms. The results showed significant increases of GAL protein levels in the peri-infarct region (P) and infarct core (I) within 48 h R of MCAO mice (p<0.001). The RT-qPCR results also demonstrated significant increases of GAL mRNA during 24-48 h R (p<0.001), and GAL receptors GalR1-2 (but not 3) mRNA levels in the P region at 24 h R of MCAO mice (p<0.001). Furthermore, the significant decrease of infarct volume (p<0.05) and improved neurological outcome (p<0.001-0.05) were observed in MCAO mice following 1 h pre- or 6 h post-treatment of GAL during 1-7 d reperfusion. GalR1 was confirmed as the receptor responsible for GAL-induced neuroprotection by using GalR2/3 agonist AR-M1896 and Lentivirus-based RNAi knockdown of GalR1. GAL treatment inhibited Caspase-3 activation through the upstream initiators Capsases-8/-12 (not Caspase-9) in both P region and OGD-treated cortical neurons. Meanwhile, GAL's neuroprotective effect was not observed in cortical neurons from conventional protein kinase C (cPKC) γ knockout mice. These results suggested that exogenous GAL protects the brain from ischemic injury by inhibiting Capsase-8/12-initiated apoptosis, possibly mediated by GalR1 via the cPKCγ signaling pathway.

Cigarette smoking is one of the major risk factors for the occurrence and progression of oral squamous cell carcinoma (OSCC). Receptor-interacting protein 2 (RIP2) has been involved in mucosal immunity and homeostasis via a positive regulation of nuclear factor κB (NF-κB) transcription factor activity. Caspase-12 can bind to RIP2 and dampen mucosal immunity. However, the roles of RIP2/NF-κB and caspase-12 in OSCC induced by cigarette smoking remain unknown. Herein, we investigated the effects of cigarette smoking on the RIP2/NF-κB and caspase-12 in human OSCC tissues and OSCC cell lines (HSC-3). We first observed that RIP2 mediated NF-κB activation and caspase-12 upregulation in OSCC patients with cigarette smoking and cigarette smoke extract (CSE)-treated HSC-3 cells, respectively. Moreover, we confirmed that the downregulation of RIP2 by siRNA resulted in the reduction of caspase-12 expression and NF-κB activity in the presence of CSE treatment in vitro. In summary, our results indicated that cigarette smoking induced the activation of the RIP2/caspase-12/NF-κB axis and it played an important role in the development of OSCC. The RIP2/caspase-12/NF-κB axis could be a target for OSCC prevention and treatment in the future.

The role of endoplasmic reticulum (ER) stress has been proposed in several neurodegenerative and autoimmune diseases and may contribute to neural apoptosis. The complex role of ER stress-mediated apoptosis introduces a novel angle on multiple sclerosis (MS) research. Nevertheless, the mechanisms through which ER stress intermediates apoptosis are not entirely understood. To this aim, we examined the expression of C/EBP homologous protein (CHOP), caspase-12, and protein disulfide isomerase (PDI) in mice with experimental autoimmune encephalomyelitis (EAE). We found the upregulated expression of CHOP, caspase-12, and PDI in the brain of EAE mice in comparison to healthy mice. Furthermore, immunofluorescent dual-label staining verified elevated levels of caspase-12/CHOP in astrocytes, oligodendrocytes, and microglia in the hippocampus section of EAE mice. This study highlighted the presence of ER stress and increased activation of caspase-12 in the hippocampus of mice in response to EAE induction. Higher levels of caspase-12/CHOP in hippocampal oligodendrocytes as compared with microglia and astrocytes denote that oligodendrocytes are particularly sensitive to ER-mediated apoptosis. In conclusion, the regulation of ER stress pathway would be beneficial for the survival of oligodendrocyte.