Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration

Our plain-English summary of Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration, by W. Blum, L. Pecze, E. Felley-Bosco, et al (Respiratory Research, 2015).

The study

• Authors: W. Blum, L. Pecze, E. Felley-Bosco, B. Schwaller
• Journal: Respiratory Research
• Published: 2015;16:153
• Cited by: 19 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: The Ca(2+)-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin's likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin's putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR-/-) and wildtype (WT) mice were compared.

Methods: Primary mouse mesothelial cells from WT and CR-/- mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system.

Results: CR-/- mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical "cobblestone-like" morphology and express mesothelial markers including mesothelin. In cells from CR-/- mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR-/- cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space.

Conclusions: We hypothesize that the differences in proliferation and mobility between WT and CR-/- mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling.

In plain English

This laboratory study examined mesothelial cells (which line body cavities) from the abdomens of mice. It compared normal mice with mice bred to lack calretinin, which the authors describe as a marker for identifying epithelioid malignant mesothelioma and reactive mesothelium. No asbestos was used in the experiments. Mice lacking calretinin had a normal-looking mesothelium. In culture, however, their cells covered on average a 74.6% larger area (excluding giant cells), grew more slowly, and closed an artificial scratch more slowly (13.7 micrometres per hour, versus 19.3 for normal cells). Over 24 hours, 73% of normal cells divided, versus 3% of calretinin-lacking cells (30 cells each). Cell death did not differ significantly. The authors attributed the slower growth to a longer G0/G1 phase of the cell cycle. Adding extra calretinin, including a nucleus-targeted form, increased growth and scratch closure in both kinds of cells. Lung fibroblasts from the two kinds of mice did not differ in growth measures or overall shape. In mouse embryos, calretinin appeared temporarily in developing mesothelial cells. The authors hypothesized that the differences arise during embryonic development.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment on primary mesothelial cells from calretinin knockout and wild-type mice, with lentiviral overexpression of calretinin, plus tissue staining of mice and mouse embryos
• Who was studied: Peritoneal mesothelial cells from 4 to 6 month old wild-type C57Bl/6J mice and calretinin knockout (CR−/−) mice. The experiments were approved by the animal care committee of the Canton of Fribourg, Switzerland. The article does not state the number of mice. Primary lung fibroblasts and embryos at embryonic days 14.5 and 16.5 were also examined.
• Exposure: No asbestos exposure was studied. The experimental factor was calretinin: either its absence (knockout mice) or its overexpression, as full-length calretinin or as a nuclear-targeted variant (NLS-CR), using a lentiviral system.
• Main results:
  • The mesothelium of calretinin knockout mice showed no abnormalities compared with wild-type mice.
  • In culture, the average surface area covered by a typical calretinin knockout cell was 74.6% larger than that of a wild-type cell. Giant cells were excluded from this analysis, which measured 25 knockout and 24 wild-type cells (p less than 0.01).
  • Wild-type cells reached 100% confluence at about 50 hours, when knockout cells from parallel cultures were at about 30% confluence. The MTT signal at 96 hours was significantly smaller in knockout cells (3 experiments, p less than 0.005).
  • Primary lung fibroblasts from wild-type and knockout mice showed no differences in growth curves or MTT values.
  • The overall morphology of lung fibroblasts did not differ between the two genotypes (data not shown).
  • Scratch closure rates were 19.3 plus or minus 4.1 micrometres per hour for wild-type cells and 13.7 plus or minus 4.2 for knockout cells (mean plus or minus standard deviation; 8 to 10 experiments per genotype; p less than 0.05). Knockout cells also moved significantly less (10 cells per genotype, p less than 0.05).
  • During a 24-hour period, 73% of wild-type cells underwent at least one mitosis, compared with 3% of knockout cells (30 cells per genotype). The S/G2/M phase lasted about 8 hours in both genotypes.
  • The density of dying cells did not differ significantly between wild-type and knockout cultures.
  • In late-passage wild-type cells, calretinin overexpression led to 80% confluence at 140 hours, compared with about 40% for control cells. Calretinin overexpression also significantly increased proliferation in knockout cells.
  • Both full-length and nuclear-targeted calretinin significantly increased scratch closure rates in cells of both genotypes, and the size of the increase was the same for the two forms.
  • Calretinin was not detected in cultured primary mesothelial cells of either genotype by Western blot or immunohistochemistry.
  • In mouse embryos, strong calretinin staining was seen in developing mesothelium made up of cuboidal cells.
• The authors' conclusion: The authors hypothesized that the differences in proliferation and mobility between wild-type and knockout mesothelial cells likely result from differences in how the cells developed. In their view, the lack of temporary calretinin expression in the embryo causes irreversible changes in growth characteristics. They stated that future experiments are needed to learn whether the absence of calretinin affects mesothelioma development in live mice.
• Limitations the authors state:
  • The precise length of the G1 phase could not be determined, because many cells, particularly knockout cells, did not divide during the observation period.
  • In cultured wild-type cells, calretinin was either absent or below the detection limit of the methods used.
  • Growth of primary cells varied between cultures, so growth curves were compared side by side rather than in absolute terms.
  • The scratch assay only partly mimics injury to the mesothelium in the body.
  • Overexpressing calretinin in knockout cells did not fully restore wild-type cell morphology.

Topics

Medical terms in this study, explained in our Lexicon: Calretinin, Mesothelial Cells, Epithelioid, Malignant Mesothelioma, Mesothelium, Mesothelioma, Mesothelin.

Related studies

• Calretinin is essential for mesothelioma cell growth/survival in vitro: a potential new target for malignant mesothelioma therapy? — W. Blum, B. Schwaller (2013)
• The Wnt regulator SFRP4 inhibits mesothelioma cell proliferation, migration, and antagonizes Wnt3a via its netrin-like domain — V. Perumal, A.M. Dharmarajan, et al. (2017)
• SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis — T. Henzi, W.V. Blum, et al. (2009)
• Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin — J. Wörthmüller, V. Salicio, et al. (2019)
• LRRN4 and UPK3B are markers of primary mesothelial cells — M. Kanamori-Katayama, A. Kaiho, et al. (2011)
• Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation — A.L. Sherwood, S.E. Mutsaers, et al. (2008)

Browse return to medical research.