Ubiquitous Calpains Promote Caspase-12 and JNK Activation during Endoplasmic Reticulum Stress-induced Apoptosis

Abstract

Ubiquitously expressed -and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of -and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.Calpains are a family of Ca 2ϩ -dependent intracellular cysteine proteases. By cleaving their protein substrates, ubiquitously expressed -calpain and m-calpain are implicated in a wide variety of biological functions including cell migration, cell cycle regulation, differentiation, and apoptosis (reviewed in Ref. 1). Both -and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes capn1 and capn2, respectively, and a common small 28-kDa regulatory subunit encoded by the capn4 gene. The small subunit is essential to calpain activities, as shown by in vitro biochemical studies where a 25-amino acid truncation at the C terminus abolished all detectable calpain activity (2). This provided the rationale for the first reported capn4 knock-out mouse model, which interrupted the coding sequences in exon 9 (3, 4) and was predicted to truncate 38 C-terminal amino acids from the small subunit. The resulting hypothetical small subunit was not detectable, the steady-state levels of -80, m-80 catalytic subunits were reduced, and no calpain activity was observed (4). More recently, we have developed a conditionally targeted capn4 locus by inserting loxP sites into intron 8 and the noncoding region of exon 11.2 This allows conditional knock-out of capn4 by Cre-mediated recombination. Although a hypothetical small subunit protein with a 60-amino acid deletion at the C terminus might still be produced, no detectable small subunit was detected, probably because of destabilization of the hypothetical truncated protein. Expression of the large subunits was also greatly diminished, supporting the proposed role for the small subunit in stabilizing large subunits. As expected, mouse embryonic fibroblasts (MEFs) 3 from these knockouts also lacked any detectable ubiquitous calpain activity.2 Many reports on calpain function are based on using small molecule inhibitors, which lack specificity. In contrast, this genetic knock-out model is completely selective, and therefore provides a powerful tool to address the physiological functions of the ubiquitous calpains.

Reversibility of fibrosis, inflammation, and endoplasmic reticulum stress in the liver of rats fed a methionine–choline-deficient diet

“…57 Activated caspase-12 then cleaves and activates procaspase-9, which in turn activates the downstream caspase cascade, including caspase-3, DNA fragmentation, and cell death. 58 Our results also showed that protein and gene expressions of caspase-12, caspase-7, and cleaved caspase-7 were significantly elevated during steatohepatitis together with the increased number of apoptotic hepatocytes, whereas they were reduced markedly after changing the diet. Furthermore, although the proliferation of hepatocytes did not show a clear change in the MCDD group, the ratio of hepatocyte proliferation/apoptosis decreased significantly, whereas it increased markedly after changing the diet to CD, indicating that the apoptotic caspase-12 pathway may also inhibit hepatocyte proliferation in MCDD-induced steatohepatitis.…”

BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation

“…The negative results represented in Figure 2c led us to analyze other possibilities for calpainmediated BAX activation. Two manuscripts have already described that calpains control JNK1 activation 20 and that AIF is released from mitochondria by JNK1-mediated activation. 7 Moreover, it has been demonstrated that JNK phosphorylation and activation of the BH3-only proteins, BIM and BAD, couple the stress-activated signaling pathway to the BAX-dependent cell death machinery.…”

Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats

“…The unfolded and misfolded proteins activate a self-protective mechanism to upregulate the expression of some ER resident chaperones, such as glucose regulated protein 78 (GRP78), giving the affected cells a chance to survive (Reddy et al 2003;Luo et al 2006). When the cells are exposed to prolonged or strong ER stress, however, the expression of some apoptosis promoters, such as growth arrest and DNA damage-inducible gene 153 (GADD153), increases (Benavides et al 2005;McCullough et al 2001), caspase-12 is activated and in turn activates caspase-9 and caspase-3 (Morishima et al 2002;Tan et al 2006), resulting in apoptotic cell death. Because ER stress is so crucial to cell survival or death, it has become an attractive research focus in recent years.…”