calpastatin and Inflammation

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Topics: 3T3 Cells; Adipocytes; Adipose Tissue; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Collagen; Diet, High-Fat; Disease Models, Animal; Fibrosis; Inflammation; Liver; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Obesity; Weight Gain

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice.. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII.. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Topics: Angiotensin II; Animals; Aortic Aneurysm, Abdominal; Atherosclerosis; Bone Marrow Transplantation; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Movement; Cells, Cultured; Coculture Techniques; Cysteine Proteinase Inhibitors; Diet, High-Fat; Disease Models, Animal; Endothelial Cells; Genetic Predisposition to Disease; Inflammation; Leukocytes; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phenotype; Receptors, LDL; Whole-Body Irradiation

Viral myocarditis (VMC) treatment has long been lacking of effective methods. Our former studies indicated roles of calpain in VMC pathogenesis. This study aimed at verifying the potential of calpain in Coxsackievirus B3 (CVB3)-induced myocarditis treatment.. A transgenic mouse overexpressing the endogenous calpain inhibitor, calpastatin, was introduced in the study. VMC mouse model was established via intraperitoneal injection of CVB3 in transgenic and wild mouse respectively. Myocardial injury was assayed histologically (HE staining and pathology grading) and serologically (myocardial damage markers of CK-MB and cTnI). CVB3 replication was observed in vivo and in vitro via the capsid protein VP1 detection or virus titration. Inflammation/fibrotic factors of MPO, perforin, IFNγ, IL17, Smad3 and MMP2 were evaluated using western blot or immunohistology stain. Role of calpain in regulating fibroblast migration was studied in scratch assays.. Calpastatin overexpression ameliorated myocardial injury induced by CVB3 infection significantly in transgenic mouse indicated by reduced peripheral CK-MB and cTnI levels and improved histology injury. Comparing with CVB3-infected wild type mouse, the transgenic mouse heart tissue carried lower virus load. The inflammation factors of MPO, perforin, IFNγ and IL17 were down-regulated accompanied with fibrotic agents of Smad3 and MMP2 inhibition. And calpain participated in the migration of fibroblasts in vitro, which further proves its role in regulating fibrosis.. Calpain plays dual roles of facilitating CVB3 replication and inflammation promotion. Calpain inhibition in CVB3-induced myocarditis showed significant treatment effect. Calpain might be a novel target for VMC treatment in clinical practices.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Migration Inhibition; Coxsackievirus Infections; Disease Models, Animal; Enterovirus B, Human; Fibroblasts; Fibrosis; Inflammation; Mice; Myocarditis; Virus Replication

While multiple molecular mechanisms contribute to midbrain nigrostriatal dopaminergic degeneration in Parkinson's disease (PD), the mechanism of damage in non-dopaminergic sites within the central nervous system, including the spinal cord, is not well-understood. Thus, to understand the comprehensive pathophysiology underlying this devastating disease, postmortem spinal cord tissue samples (cervical, thoracic, and lumbar segments) from patients with PD were analyzed compared to age-matched normal subjects or Alzheimer's disease for selective molecular markers of neurodegeneration and inflammation. Distal axonal degeneration, relative abundance of both sensory and motor neuron death, selective loss of ChAT(+) motoneurons, reactive astrogliosis, microgliosis, increased cycloxygenase-2 (Cox-2) expression, and infiltration of T cells were observed in spinal cord of PD patients compared to normal subjects. Biochemical analyses of spinal cord tissues revealed associated inflammatory and proteolytic events (elevated levels of Cox-2, expression and activity of μ- and m-calpain, degradation of axonal neurofilament protein, and concomitantly low levels of endogenous inhibitor - calpastatin) in spinal cord of PD patients. Thus, pathologically upregulated calpain activity in spinal cords of patients with PD may contribute to inflammatory response-mediated neuronal death, leading to motor dysfunction. We proposed calpain over-activation and calpain-calpastatin dysregulation driving in a cascade of inflammatory responses (microglial activation and T cell infiltration) and degenerative pathways culminating in axonal degeneration and neuronal death in spinal cord of Parkinson's disease patients. This may be one of the crucial mechanisms in the degenerative process.

Topics: Alzheimer Disease; Axons; Calcium-Binding Proteins; Calpain; Case-Control Studies; Cell Death; Cytoskeletal Proteins; Gliosis; Humans; Huntington Disease; Inflammation; Multiple Sclerosis; Nerve Degeneration; Neurons; Parkinson Disease; Spinal Cord; T-Lymphocytes

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

Topics: Actins; Animals; Blood Vessels; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Differentiation; Cell Movement; Cells, Cultured; Cicatrix; Collagen; Female; Fibroblasts; Fibrosis; Granulation Tissue; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myofibroblasts; Platelet Endothelial Cell Adhesion Molecule-1; Skin; Wound Healing

The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential proinflammatory molecule in the kidney, given a recent report that it is an alternative urokinase plasminogen activator (uPA) receptor, in addition to the classical receptor uPAR. Two animal models and in vitro monocyte studies were involved: (1) In an ApoE(-/-) mouse model of chronic kidney disease, glomerular-resident cells and monocytes/macrophages were identified as the primary cell types that express nAChRα1 during hypercholesterolemia/uninephrectomy-induced nephropathy. Silencing of the nAChRα1 gene for 4 months (6 months on Western diet) prevented the increases in renal monocyte chemoattractant protein-1 and osteopontin expression levels and F4/80+ macrophage infiltration compared with the nonsilenced mice. These changes were associated with significantly reduced transforming growth factor-β1 mRNA (50% decrease) and α smooth muscle actin-positive (αSMA+) myofibroblasts (90% decrease), better glomerular and tubular basement membranes (GBM/TBM) preservation (threefold less disintegration), and better renal function preservation (serum creatinine 40% lower) in the nAChRα1-silenced mice. The nAChRα1 silencing was also associated with significantly reduced renal tissue calcium deposition (78% decrease) and calpain-1 (but not calpain-2) activation (70% decrease). (2) The nAChRα1 was expressed in vitro by mouse monocyte cell line WEHI-274.1. The silencing of nAChRα1 significantly reduced both calpain-1 and -2 activities, and reduced the degradation of the calpain substrate talin. (3) To further explore the role of calpain-1 activity in hypercholesterolemic nephropathy, disease severities were compared in CAST(-/-)ApoE(-/-) (calpain overactive) mice and ApoE(-/-) mice fed with Western diet for 10 months (n=12). Macrophages were the main cell type of renal calpain-1 production in the model. The number of renal F4/80+ macrophages was 10-fold higher in the CAST(-/-)ApoE(-/-) mice (P<0.05), and was associated with a significantly higher level of αSMA+ cells, increased GBM/TBM destruction, and higher serum creatinine levels. Our studies suggest that the receptor nAChRα1 is an important regulator of calpain-1 activation and inflammation in the chronic hypercholesterolemic nephropathy. This new proinflammatory pathway may also be relevant to other disorders beyond hyperlipidemic nephropathy.

Topics: Actins; Animals; Antigens, Differentiation; Apolipoproteins E; Blotting, Northern; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Line; Female; Hypercholesterolemia; Inflammation; Kidney; Kidney Diseases; Macrophages; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Nephrectomy; Receptors, Nicotinic; RNA Interference; Transforming Growth Factor beta1

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.

Topics: Arthritis, Rheumatoid; Calcium-Binding Proteins; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Humans; Inflammation; Interleukin-17; Interleukin-6; NF-kappa B; Protein Conformation; Signal Transduction; STAT5 Transcription Factor; Th17 Cells

Glioblastoma is the deadliest brain tumor that remains incurable. We examined efficacy of combination of retinoid and interferon-gamma (IFN-gamma) in human glioblastoma T98G and U87MG cells. We conjectured that retinoid could induce differentiation with down regulation of telomerase activity to increase sensitivity to IFN-gamma for apoptosis in glioblastoma cells. Indeed, treatment of cells with 1 muM all-trans retinoic acid (ATRA) or 1 muM 13-cis retinoic acid (13-CRA) for 7 days induced astrocytic differentiation with upregulation of glial fibrillary acidic protein (GFAP) and down regulation of telomerase activity. Wright staining and ApopTag assay showed, respectively, morphological and biochemical features of apoptosis in glioblastoma cells following exposure to 200 units/ml IFN-gamma for 48 h. Induction of differentiation was associated with decreases in levels of nuclear factor kappa B (NFkappaB), inducible nitric oxide synthase (iNOS), and production of nitric oxide (NO) so as to increase sensitivity to IFN-gamma for apoptosis. Notably, IFN-gamma induced signal transducer and activator of transcription-1 (STAT-1) to bind to gamma-activated sequence (GAS) of the target gene. Also, IFN-gamma activated caspase-8 and cleaved Bid to truncated Bid (tBid) for translocation to mitochondria. Fura-2 assay showed increases in intracellular free [Ca2+] and activation of calpain in apoptotic cells. Besides, increases in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and Smac into the cytosol activated caspase-9 and caspase-3 for apoptosis. Taken together, our results showed that retinoid induced astrocytic differentiation with down regulation of telomerase activity and enhanced sensitivity to IFN-gamma for increasing apoptosis in human glioblastoma cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Calcium-Binding Proteins; Caspase 1; Caspase 8; Cell Differentiation; Down-Regulation; Glial Fibrillary Acidic Protein; Glioblastoma; Humans; Inflammation; Interferon-gamma; Intracellular Signaling Peptides and Proteins; Isotretinoin; Mitochondria; Mitochondrial Proteins; Nitric Oxide Synthase Type II; Retinoids; Telomerase; Tretinoin; Tumor Cells, Cultured; Up-Regulation

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Topics: Angiotensin II; Animals; Aorta; Blood Pressure; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fibrosis; Genetic Therapy; Hypertension; Hypertrophy; Hypertrophy, Left Ventricular; Inflammation; Infusion Pumps, Implantable; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocardium; NF-kappa B; NFATC Transcription Factors; Renal Artery; Time Factors; Ventricular Remodeling

Glomerular injury and albuminuria in acute glomerulonephritis are related to the severity of inflammatory process. Calpain, a calcium-activated cysteine protease, has been shown to participate in the development of the inflammatory process. Therefore, for determination of the role of calpain in the pathophysiology of acute glomerulonephritis, transgenic mice that constitutively express high levels of calpastatin, a calpain-specific inhibitor protein, were generated. Wild-type mice that were subjected to anti-glomerular basement membrane nephritis exhibited elevated levels of calpain activity in kidney cortex at the heterologous phase of the disease. This was associated with the appearance in urine of calpain activity, which originated potentially from inflammatory cells, abnormal transglomerular passage of plasma proteins, and tubular secretion. In comparison with nephritic wild-type mice, nephritic calpastatin-transgenic mice exhibited limited activation of calpain in kidney cortex and limited secretion of calpain activity in urine. This was associated with less severe glomerular injury (including capillary thrombi and neutrophil activity) and proteinuria. There was a reduction in NF-kappaB activation, suggesting that calpain may participate in inflammatory lesions through NF-kappaB activation. There also was a reduction in nephrin disappearance from the surface of podocytes, indicating that calpain activity would enhance proteinuria by affecting nephrin expression. Exposure of cultured podocytes to calpain decreased nephrin expression, and, conversely, exposure of these cells to calpastatin prevented TNF-alpha from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury.

Topics: Albuminuria; Animals; Anti-Glomerular Basement Membrane Disease; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Female; Glomerulonephritis; Inflammation; Kidney; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B