Abstract
BackgroundThe calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.MethodsWe searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.ResultsSeptin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.ConclusionsOur results demonstrate septin 7 not only serving as a “cytoskeletal” protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.
Highlights
The calcium-binding protein calretinin is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM)
Since MM cell lines of the epithelioid and biphasic type were previously shown to be strongly affected by CR down-regulation, much more than MM cells derived from sarcomatoid tumors [3], further experiments were carried out with ZL5 and ZL55 and MSTO-211H cells
Calretinin and septin 7 are co-expressed during mouse embryonic development and septin 7 levels are higher in primary mesothelial cells from mice without a functional Calb2 gene Based on previous findings that CR is expressed in specific regions within the mesenchyme of murine embryos and in precursor mesothelial cells in the developing lung at embryonic days 14.5 and 16.5 [25], we investigated the expression of CR and septin 7 on serial sections derived from mouse embryos at E10.5
Summary
The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). Downregulation of CR by CALB2 shRNA in human MM cell lines profoundly decreases cell growth and viability in vitro: lentivirus-mediated delivery of shCALB2 causes MM cells, in particular the ones with an epithelioid morphology, to enter apoptosis within 72 h post-infection [3]. Under these conditions, the intrinsic caspase 9dependent pathway is activated. Neither is the viability impaired nor any type of cell death pathway activated
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