Visualizing cell death in live retina: Using calpain activity detection as a biomarker for retinal degeneration

Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the t-BOC-Leu-Met-CMAC calpain-specific substrate to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and RhoP23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with an immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD, suitable for combination with non-invasive imaging techniques.

Insulin receptor cleavage induced by estrogen impairs insulin signaling

IntroductionSoluble insulin receptor (sIR), which is the ectodomain of insulin receptor (IR), is present in human plasma. Plasma sIR levels are positively correlated with blood glucose levels and negatively correlated with insulin sensitivity. An in vitro model of IR cleavage shows that extracellular calpain 2 directly cleaves IR, which generates sIR, and sequential cleavage of the IRβ subunit by γ-secretase impairs insulin signaling in a glucose concentration-dependent manner. Nevertheless, sIR levels vary among subjects with normal glucose levels.Research design and methodsWe examined sIR levels of pregnant women throughout gestation. Using an in vitro model, we also investigated the molecular mechanisms of IR cleavage induced by estradiol.ResultsIn pregnant women, sIR levels were positively correlated with estrogen levels and significantly increased at late pregnancy independent of glucose levels. Using an in vitro model, estrogen elicited IR cleavage and impaired cellular insulin signaling. Estradiol-induced IR cleavage was inhibited by targeting of calpain 2 and γ-secretase. Estrogen exerted these biological effects via G protein-coupled estrogen receptor, and its selective ligand upregulated calpain 2 expression and promoted exosome secretion, which significantly increased extracellular calpain 2. Simultaneous stimulation of estrogen and high glucose levels had a synergic effect on IR cleavage. Metformin prevented calpain 2 release in exosomes and restored insulin signaling impaired by estrogen.ConclusionsEstradiol-induced IR cleavage causes cellular insulin resistance, and its molecular mechanisms are shared with those by high glucose levels. sIR levels at late pregnancy are significantly elevated along with estrogen levels. Therefore, estradiol-induced IR cleavage is preserved in pregnant women and could be part of the etiology of insulin resistance in gestational diabetes mellitus and overt diabetes during pregnancy.

Tunicamycin Induces Hepatic Stellate Cell Apoptosis Through Calpain-2/Ca2 +-Dependent Endoplasmic Reticulum Stress Pathway

It has been reported that calpain/caspase-mediated apoptosis induced by endoplasmic reticulum stress (ERS) in hepatic stellate cells (HSCs) by previous studies. At present, the activation of HSC is an important cause of liver fibrosis, and the induction of HSC apoptosis plays an irreplaceable role in reversing liver fibrosis. Therefore, it is of great significance to explore mechanisms of action that can induce HSC apoptosis for the reversal of hepatic fibrosis and the clinical prevention and treatment of hepatic-fibrosis-related diseases such as hepatitis, cirrhosis, and liver cancer. In the current study, we demonstrated that tunicamycin (a novel ERS inducer) can induce the apoptosis of HSCs and increase the concentration of intracellular Ca2+ and the expression of ERS protein GRP78, apoptosis protein caspase-12, and Bax, while it can decrease the antiapoptosis protein expression of Bcl-2. Our findings indicate that tunicamycin can induce HSCs apoptosis through calpain-2/Ca2+-dependent ERS pathway.

Calpain-2 Specifically Cleaves Junctophilin-2 at the Same Site as Calpain-1 but with Less Efficacy

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining excitation-contraction coupling. We previously demonstrated that JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75-kD N-terminal (JP2NT) and 25-kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25-kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35-kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and β2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.

MicroRNA-137 Inhibited Hypoxia-Induced Proliferation of Pulmonary Artery Smooth Muscle Cells by Targeting Calpain-2

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodeling in pulmonary hypertension (PH). It has been reported that miR-137 inhibits the proliferation of tumor cells. However, whether miR-137 is involved in PH remains unclear. In this study, male Sprague-Dawley rats were subjected to 10% O2 for 3 weeks to establish PH, and rat primary PASMCs were treated with hypoxia (3% O2) for 48 h to induce cell proliferation. The effect of miR-137 on PASMC proliferation and calpain-2 expression was assessed by transfecting miR-137 mimic and inhibitor. The effect of calpain-2 on PASMC proliferation was assessed by transfecting calpain-2 siRNA. The present study found for the first time that miR-137 was downregulated in pulmonary arteries of hypoxic PH rats and in hypoxia-treated PASMCs. miR-137 mimic inhibited hypoxia-induced PASMC proliferation and upregulation of calpain-2 expression in PASMCs. Furthermore, miR-137 inhibitor induced the proliferation of PASMCs under normoxia, and knockdown of calpain-2 mRNA by siRNA significantly inhibited hypoxia-induced proliferation of PASMCs. Our study demonstrated that hypoxia-induced downregulation of miR-137 expression promoted the proliferation of PASMCs by targeting calpain-2, thereby potentially resulting in pulmonary vascular remodeling in hypoxic PH.

Cleavage and activation of LIM kinase 1 as a novel mechanism for calpain 2-mediated regulation of nuclear dynamics

Calpain-2 (CAPN2) is a processing enzyme ubiquitously expressed in mammalian tissues whose pleiotropic functions depend on the role played by its cleaved-products. Nuclear interaction networks, crucial for a number of molecular processes, could be modified by CAPN2 activity. However, CAPN2 functions in cell nucleus are poorly understood. To unveil CAPN2 functions in this compartment, the result of CAPN2-mediated interactions in cell nuclei was studied in breast cancer cell (BCC) lines. CAPN2 abundance was found to be determinant for its nucleolar localization during interphase. Those CAPN2-dependent components of nucleolar proteome, including the actin-severing protein cofilin-1 (CFL1), were identified by proteomic approaches. CAPN2 binding, cleavage and activation of LIM Kinase-1 (LIMK1), followed by CFL1 phosphorylation was studied. Upon CAPN2-depletion, full-length LIMK1 levels increased and CFL1/LIMK1 binding was inhibited. In addition, LIMK1 accumulated at the cell periphery and perinucleolar region and, the mitosis-specific increase of CFL1 phosphorylation and localization was altered, leading to aberrant mitosis and cell multinucleation. These findings uncover a mechanism for the role of CAPN2 during mitosis, unveil the critical role of CAPN2 in the interactions among nuclear components and, identifying LIMK1 as a new CAPN2-target, provide a novel mechanism for LIMK1 activation. CFL1 is crucial for cytoskeleton remodeling and mitosis, but also for the maintenance of nuclear structure, the movement of chromosomes and the modulation of transcription frequently altered in cancer cells. Consequently, the role of CAPN2 in the nuclear compartment might be extended to other actin-associated biological and pathological processes.