The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The -and m-calpains consist of a -or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4 ؊/؊ mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4؊/؊ cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4 ؊/؊ cells, but the cells had prominent vinculincontaining focal complexes at the cell periphery. The levels of the focal adhesion proteins, ␣-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4 ؉/؉ and Capn4 ؊/؊ cells. FAK, ␣-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4 ؊/؊ cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4 ؊/؊ embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.

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.

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.

Moffatt Christine, Kommala Dheerendra, Dourdin Nathalie, Choe Yoonhee. Venous leg ulcers: patient concordance with compression therapy and its impact on healing and prevention of recurrence. ABSTRACT This study aimed to review available data on the reasons attributed to patient non concordance with compression therapy for the treatment of venous leg ulcers (VLUs), the frequency of non concordance and its effects on clinical outcomes. The biomedical literature was searched for publications on VLUs, compression therapy and concordance over the past 20 years. Physical, aesthetic and cosmetic factors, patient lack of education about VLUs, cost of therapy and issues with treatment by clinicians were all reported to influence concordance with compression therapy. The search identified 10 studies reporting patient concordance with compression stockings or bandages; while non concordance ranged from 2% to 42% of patients in three randomised controlled trials, it was generally higher in real-world studies, ranging from 9.7% to 80%. Another set of six studies indicated that the healing rate was half and the median time to complete healing was twice as long when patients were not concordant. Further, recurrence rates were 2-20 times greater when patients did not comply with the use of stockings following VLU healing. In conclusion, published biomedical literature has documented that non concordance with compression therapy negatively impacts the outcome of VLUs, highlighting the need to improve patient concordance to maximise therapeutic benefits.

Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways.Genetic alterations in the normal mammary epithelium, including the activation of oncogenes and the loss of function of tumor suppressor genes, have been implicated in the induction of mammary tumors. ErbB-2 (Neu/HER2) is a member of the epidermal growth factor receptor (EGFR) 4 family of receptor tyrosine kinases, which also includes EGFR, ErbB-3 (HER3), and ErbB-4 (HER4) (1). Amplification/overexpression of ErbB-2 has been observed in 20 -30% of human breast cancers, correlating with poor patient prognosis (2, 3). The importance of ErbB-2 in mammary tumorigenesis has been demonstrated by several transgenic mouse models (4), where engagement of the ErbB-2 receptor results in the recruitment of adaptor proteins, activating primarily the Ras pathway (5). ErbB-2-induced mammary tumors exhibit elevated ErbB-3 protein levels (6). ErbB-3 mediates activation of the phosphatidylinositol 3Ј-kinase (PI3K)/Akt pathway by recruiting the p85 regulatory subunit of PI3K (7,8). Additionally, the ErbB-2/ErbB-3 heterodimer is thought to be the most biologically active and protumorigenic receptor complex (9, 10). These findings suggest that the concerted activation of both Ras and PI3K signaling through this EGFR family heterodimer plays a critical role in mammary tumorigenesis (9, 10).Growth and survival pathways can also become oncogenic through the disruption of tumor suppressors. Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) due to mutation, loss of heterozygosity (LOH), and epigenetic down-modulation has been reported in about 50% of human cancers, including br...