Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.
- Klíčová slova
- Actin, Adherens junctions, Calpains, Cell scattering, ERK, Epithelial polarity, Focal adhesions, HGF/SF, Migration, Proteases, Tight junctions, Transepithelial electrical resistance,
- MeSH
- beta-katenin metabolismus MeSH
- buněčná adheze * MeSH
- buňky MDCK MeSH
- CRISPR-Cas systémy MeSH
- epitelové buňky * metabolismus cytologie MeSH
- hepatocytární růstový faktor metabolismus MeSH
- kadheriny metabolismus MeSH
- kalpain * metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 1 metabolismus MeSH
- pohyb buněk MeSH
- proteolýza MeSH
- psi MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- beta-katenin MeSH
- hepatocytární růstový faktor MeSH
- kadheriny MeSH
- kalpain * MeSH
- mitogenem aktivovaná proteinkinasa 1 MeSH
- vápník MeSH
The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.
- Klíčová slova
- Cell scattering, Cell-cell adhesions, ERK, Epithelial plasticity, Fra1, HGF/SF,
- MeSH
- adenokarcinom * metabolismus MeSH
- buněčná adheze MeSH
- epitelové buňky metabolismus MeSH
- fosforylace MeSH
- lidé MeSH
- MAP kinasový signální systém fyziologie MeSH
- mitogenem aktivovaná proteinkinasa 1 MeSH
- mitogenem aktivované proteinkinasy kinas metabolismus MeSH
- nádory slinivky břišní * metabolismus MeSH
- proliferace buněk MeSH
- signální transdukce MeSH
- transkripční faktory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- MAPK1 protein, human MeSH Prohlížeč
- mitogenem aktivovaná proteinkinasa 1 MeSH
- mitogenem aktivované proteinkinasy kinas MeSH
- transkripční faktory MeSH
Aberrant regulation of the cell cycle is a typical feature of all forms of cancer. In head and neck squamous cell carcinoma (HNSCC), it is often associated with the overexpression of cyclin D1 (CCND1). However, it remains unclear how CCND1 expression changes between tumor and normal tissues and whether human papillomavirus (HPV) affects differential CCND1 expression. Here, we evaluated the expression of D-type cyclins in a cohort of 94 HNSCC patients of which 82 were subjected to whole genome expression profiling of primary tumors and paired normal mucosa. Comparative analysis of paired samples showed that CCND1 was upregulated in 18% of HNSCC tumors. Counterintuitively, CCND1 was downregulated in 23% of carcinomas, more frequently in HPV-positive samples. There was no correlation between the change in D-type cyclin expression and patient survival. Intriguingly, among the tumors with downregulated CCND1, one-third showed an increase in cyclin D2 (CCND2) expression. On the other hand, one-third of tumors with upregulated CCND1 showed a decrease in CCND2. Collectively, we have shown that CCND1 was frequently downregulated in HNSCC tumors. Furthermore, regardless of the HPV status, our data suggested that a change in CCND1 expression was alleviated by a compensatory change in CCND2 expression.
- Klíčová slova
- 11q13 amplification, CCND1, CCND2, CCND3, D-type cyclins, cell cycle, head and neck squamous cell carcinoma, human papillomavirus, paired tumor-normal samples, patient survival,
- Publikační typ
- časopisecké články MeSH
