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Polymerase chain reaction and focal contact formation indicate integrin expression in mesangial cells
1Nephrology Research Laboratory, Medizinische Klinik IV, University of Erlangen-Nürnberg, Germany.
Abstract:
Cultured kidney glomerular mesangial cells (MCs) allow the role of extracellular matrix (ECM) and growth factors in glomerular inflammatory disease to be studied. To investigate the potential of MCs to interact with matrix components, the expression of integrin mRNA in cultured MCs was examined by polymerase chain reaction (PCR), by Northern blotting and by immunofluorescence. In addition, the effect of matrix substrates on mRNA expression was assessed by PCR. Northern blots with cDNA probes to integrin alpha-chains revealed that MCs expressed alpha 1, alpha 3 and alpha 5 integrin mRNA. alpha 1 and alpha 3 were the major messages. No alpha 2, alpha 4 or alpha 6 were detectable. RT-PCR revealed that alpha 2 and alpha 6 were also expressed at low levels. The control cells, HT1080, expressed alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 mRNA, and Rugli expressed alpha 1, alpha 3 and alpha 5, supporting previous studies. Immunocytochemistry confirmed that alpha 1 beta 1, alpha 2 beta 1, and alpha 5 beta 1 integrins were expressed and that they were concentrated into focal adhesions (alpha 1 beta 1 on type I collagen and laminin; alpha 2 beta 1 on type I collagen; alpha 3 beta 1 on type I collagen, laminin and fibronectin; alpha 5 beta 1 on fibronectin). alpha 6 beta 1 was not detected in focal contacts. Attachment, spreading, and formation of talin and integrin containing focal contacts still occurred when endogenous protein synthesis was blocked with 30 micrograms.ml-1 cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Kidney glomerular mesangial cells express various integrin receptors, crucial for interacting with the extracellular matrix. These integrins mediate cell attachment and spreading, even when protein synthesis is inhibited.
Area of Science:
- Cell Biology
- Nephrology
- Extracellular Matrix Biology
Background:
- Glomerular inflammatory diseases involve extracellular matrix (ECM) and growth factors.
- Kidney glomerular mesangial cells (MCs) are key in studying these diseases.
- Understanding MCs' interaction with ECM components is crucial.
Purpose of the Study:
- To investigate the expression of integrin mRNA in cultured MCs.
- To determine the effect of matrix substrates on integrin mRNA expression.
- To confirm integrin protein expression and localization.
Main Methods:
- Polymerase chain reaction (PCR) for mRNA detection.
- Northern blotting for mRNA analysis.
- Immunofluorescence and immunocytochemistry for protein localization.
Main Results:
- MCs express alpha 1, alpha 3, and alpha 5 integrin mRNA, with alpha 1 and alpha 3 being major.
- Low levels of alpha 2 and alpha 6 integrin mRNA were detected by RT-PCR.
- Immunocytochemistry confirmed expression of alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins in focal adhesions.
Conclusions:
- Cultured MCs express specific integrin subunits that mediate interactions with ECM proteins.
- Integrin-mediated cell adhesion and spreading are independent of de novo protein synthesis.
- These findings provide insights into the role of integrins in glomerular cell function and disease.