Calpastatin-Mediated Inhibition of Calpain Ameliorates Skin Scar Formation after Burn Injury.
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ABSTRACT:
SUBMITTER: Seo CH
PROVIDER: S-EPMC8199077 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background:The molecular pattern of severe burn-induced acute lung injury, characterized by cell structure damage and leukocyte infiltration, remains unknown. This study aimed to determine whether calpain, a protease involved in both processes, mediates severe burn-induced acute lung injury. Methods:Rats received full-thickness scald burns covering 30% of the total body surface area, followed by instant fluid resuscitation. MDL28170 (Tocris Bioscience), an inhibitor of calpain, was given intravenously 1 h before or after the scald burn. The histological score, wet/dry weight ratio, and caspase-3 activity were examined to evaluate the degree of lung damage. Calpain activity and its source were detected by an assay kit and immunofluorescence staining. The proteolysis of membrane skeleton proteins ?-fodrin and ankyrin-B, which are substrates of calpain, was measured by Western blot. Results:Time-course studies showed that tissue damage reached a peak between 1 and 6 h post-scald burn and gradually diminished at 24 h. More importantly, calpain activity reached peak levels at 1 h and was maintained until 24 h, paralleled by lung damage to some extent. Western blot showed that the levels of the proteolyzed forms of ?-fodrin and ankyrin-B correlated well with the degree of damage. MDL28170 at a dose of 3 mg/kg b. w. given 1 h before burn injury not only antagonized the increase in calpain activity but also ameliorated scald burn-induced lung injury, including the degradation of ?-fodrin and ankyrin-B. Immunofluorescence images revealed calpain 1 and CD45 double-positive cells in the lung tissue of rats exposed to scald burn injury, suggesting that leukocytes were a dominant source of calpain. Furthermore, this change was blocked by MDL28170. Finally, MDL28170 given at 1 h post-scald burn injury significantly ameliorated the wet/dry weight ratio compared with burn injury alone. Conclusions:Calpain, a product of infiltrating leukocytes, is a mediator of scald burn-induced acute lung injury that involves enhancement of inflammation and proteolysis of membrane skeleton proteins. Its late effects warrant further study.
| S-EPMC6174571 | biostudies-literature
Project description:We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5-10% CSE for 2-24 h. eNOS gene transcription rate, eNOS messenger ribonucleic acid (mRNA) half-life, and the activity and protein contents of calpain and calpastatin were measured. Incubation of PAEC with CSE caused significant decreases in eNOS gene transcription and calpain activity and an increase in calpastatin protein content. eNOS mRNA half-life was not significantly altered by CSE. To investigate whether CSE-induced inhibition of eNOS gene expression is caused by decreased calpain activity due to an increase in calpastatin protein content, we cloned calpastatin gene from PAEC and constructed adenovirus vectors containing calpastatin. Overexpression of calpastatin mimics the inhibitory effects of CSE on calpain activity and on the activity, protein, and mRNA of eNOS. The cell-permeable calpain inhibitor, calpastatin peptide, inhibits acetylcholine-induced endothelium-dependent relaxation of the pulmonary artery. Incubation of PAEC with an antisense oligodeoxyribonucleotide of calpastatin prevented CSE-induced increases in calpastatin protein and CSE-induced decreases in calpain activity, eNOS gene transcription, activity and protein content of eNOS, and NO release. These results indicate that CSE-induced inhibition of eNOS expression in PAEC is caused by calpain inhibition due to an increase in calpastatin protein content.
| S-EPMC2715359 | biostudies-literature
Project description:Glioblastoma (GBM) is an aggressive, malignant brain tumor that inevitably develops resistance to conventional chemotherapy and radiation treatments. In order to identify signaling pathways involved in the development of radiation resistance, we performed mass spectrometry-based phospho-proteomic profiling of GBM cell lines and normal human astrocytes before and after radiation treatment. We found radiation induced phosphorylation of a number of proteins including calpastatin, specifically in GBM stem cells (GSCs). Herein, we focused on calpastatin, an endogenous inhibitor of calpain proteases. Radiation-induced phosphorylation of calpastatin at Ser-633 within the inhibitory domain was validated with a phospho-specific antibody. In order to test the functional significance of phosphorylated calpastatin, we utilized site-directed mutagenesis to generate phospho-inactive (Ser633Ala) and phospho-mimetic (Ser633Glu) mutant calpastatin. GBM cell lines stably expressing the mutant calpastatin showed that phosphorylation was necessary for radiation-induced calpain activation. We also showed that casein kinase 2, a pro-survival kinase overexpressed in many cancer types, phosphorylated calpastatin at Ser-633. Our results indicate that calpastatin phosphorylation promotes radiation resistance in GBM cells by increasing the activity of calpain proteases, which are known to promote survival and invasion in cancer.
| S-EPMC5865692 | biostudies-literature
Project description:The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.
| S-EPMC2646846 | biostudies-literature
Project description:Traumatic brain injury (TBI) causes neuronal apoptosis, inflammation, and reactive astrogliosis, which contribute to secondary tissue loss, impaired regeneration, and associated functional disabilities. Here, we show that up-regulation of cell cycle components is associated with caspase-mediated neuronal apoptosis and glial proliferation after TBI in rats. In primary neuronal and astrocyte cultures, cell cycle inhibition (including the cyclin-dependent kinase inhibitors flavopiridol, roscovitine, and olomoucine) reduced up-regulation of cell cycle proteins, limited neuronal cell death after etoposide-induced DNA damage, and attenuated astrocyte proliferation. After TBI in rats, flavopiridol reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus. Treatment also decreased neuronal cell death and lesion volume, reduced astroglial scar formation and microglial activation, and improved motor and cognitive recovery. The ability of cell cycle inhibition to decrease both neuronal cell death and reactive gliosis after experimental TBI suggests that this treatment approach may be useful clinically.
| S-EPMC1149422 | biostudies-literature
Project description:The immune response is determined by the speed of the T cell reaction to antigens assured by a state of readiness for proliferation and cytokine secretion. Proliferation, apoptosis and motion of many cell types are controlled by cytoplasmic proteases - µ- and m-calpain - and their inhibitor calpastatin, together forming the "calpain-calpastatin system" (CCS), assumed to modify their targets only upon activation-dependent cytoplasmic Ca2+ increase. Contrastingly to this notion, using quantitative real time PCR and semiquantitative flow cytometry respectively, we show here that the CCS genes are constitutively expressed, and that both calpains are constitutively active in resting, circulating human CD4+ and CD8+ lymphocytes. Furthermore, we demonstrate that calpain inhibition in the resting T cells prevents them from proliferation in vitro and greatly reduces secretion of multiple cytokines. The mechanistic reason for these effects of calpain inhibition on T cell functions might be the demonstrated significant reduction of the expression of active (phosphorylated) upstream signalling molecules, including the phospholipase C gamma, p56Lck and NFκB, in the inhibitor-treated cells. Thus, we propose that the constitutive, self-regulatory calpain-calpastatin system activity in resting human T cells is a necessary, controlling element of their readiness for complex and effective response to antigenic challenge.
| S-EPMC5363525 | biostudies-literature
Project description:A subset of human skin fibroblasts are phagocytic. To investigate the gene expression changes between phagocytosing and non-phagocytosing fibroblasts, fibroblasts were incubated with apoptotic human dermal microvascular endothelial cells expressing green fluorescent protein or no treatment. Treated fibroblasts were then collected after 8 hours and sorted by FACS to identify phagocytic and non-phagocytic fibroblasts. We then performed gene expression profiling analysis using data obtained from RNA-seq of these three groups of cells.
2023-06-01 | GSE224202 | GEO
Project description:The current standard of care for the prevention and treatment of scarring after burn injury is pressure garment therapy. Although this therapy has been used clinically for many years, controversy remains regarding its efficacy. The authors evaluated the efficacy of pressure garment therapy in a female red Duroc pig burn model in which wound depth could be tightly controlled.Full-thickness burn wounds were generated on female red Duroc pigs. At day 28 after burn, pressure garment therapy was applied to half the wounds (10 mmHg), with control wounds covered with garments that exerted no compression. Scar area, perfusion, hardness, and elasticity were quantified at days 0, 28, 42, 56, and 72 using computerized planimetry, laser Doppler, and torsional ballistometry. Scar morphology was assessed at days 28, 56, and 76 using histology, immunohistochemistry, and transmission electron microscopy.Pressure garment therapy significantly hindered scar contraction, with control scars contracting to 64.6 percent + 13.9 percent original area at day 72, whereas pressure garment therapy scars contracted to 82.7 percent + 17.9 percent original area. Pressure garments significantly reduced skin hardness and increased skin strength by 1.3 times. No difference in perfusion or blood vessel density was observed. The average collagen fiber diameter was greater in control burns than in pressure garment therapy.Pressure garment therapy was effective at reducing scar contraction and improving biomechanics compared with control scars. These results confirm the efficacy of pressure garments and highlight the need to further investigate the role of pressure magnitude and the time of therapy application to enhance efficacy for optimal biomechanics and patient mobility.
| S-EPMC4552574 | biostudies-literature
Project description:To demonstrate the pro-osteogenic effect of burn injury on heterotopic bone formation using a novel burn ossicle in vivo model.Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, is a troubling sequela of burn and trauma injuries. The exact mechanism by which burn injury influences bone formation is unknown. The aim of this study was to develop a mouse model to study the effect of burn injury on heterotopic bone formation. We hypothesized that burn injury would enhance early vascularization and subsequent bone formation of subcutaneously implanted mesenchymal stem cells.Mouse adipose-derived stem cells were harvested from C57/BL6 mice, transfected with a BMP-2 adenovirus, seeded on collagen scaffolds (ossicles), and implanted subcutaneously in the flank region of 8 adult mice. Burn and sham groups were created with exposure of 30% surface area on the dorsum to 60°C water or 30°C water for 18 seconds, respectively (n = 4/group). Heterotopic bone volume was analyzed in vivo by micro-computed tomography for 3 months. Histological analysis of vasculogenesis was performed with platelet endothelial cell adhesion molecule staining. Osteogenic histological analysis was performed by Safranin O, Picrosirius red, and aniline blue staining. Qualitative analysis of heterotopic bone composition was completed with ex vivo Raman spectroscopy.Subcutaneously implanted ossicles formed heterotopic bone. Ossicles from mice with burn injuries developed significantly more bone than sham control mice, analyzed by micro-computed tomography at 1, 2, and 3 months (P < 0.05), and had enhanced early and late endochondral ossification as demonstrated by Safranin O, Picrosirius red, and aniline blue staining. In addition, burn injury enhanced vascularization of the ossicles (P < 0.05). All ossicles demonstrated chemical composition characteristic of bone as demonstrated by Raman spectroscopy.Burn injury increases the predilection to osteogenic differentiation of ectopically implanted ossicles. Early differences in vascularity correlated with later bone development. Understanding the role of burn injury on heterotopic bone formation is an important first step toward the development of treatment strategies aimed to prevent unwanted and detrimental heterotopic bone formation.
| S-EPMC4498401 | biostudies-other
Project description:The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.
| S-EPMC1950658 | biostudies-literature