THE ROLE OF LMNA MUTATIONS IN MYOGENIC DIFFERENTIATION OF PRIMARY SATELLITE CELLS AND C2C12 CELLS.

Nuclear lamins form nuclear lamina localized under the inner nuclear membrane. It was previously considered that the nuclear lamina predominantly plays a structural role, however, its involvement have been recently described in the regulatory processes such as chromatin organization and gene transcription. It is known that mutations in the LMNA gene lead to the development of a large number of diseases, laminopathies, which mainly affect mesenchymal tissue. Nowadays, the mechanisms by which the lamina can regulate cell differentiation remain incompletely understood. In the present work, we have studied the effect of LMNA gene mutations on the process of muscle differentiation of primary satellite cells and Ñ2Ñ12 cell line. The genome of satellite cells and Ñ2Ñ12 cell line was modified by the introduction of lentiviral constructs encoding LMNA G232E associated with the development of muscular dystrophy Emery­Dreyfus and LMNA R571S associated with the development of dilated cardiomyopathy. The morphology of the cells was estimated using immunofluorescence, the expression level of myogenic genes were analyzed by qPCR. We have shown that the analyzed mutations reduce the ability of cells to differentiate, to fuse and to form myotubes. We have suggested that it is due to enhanced expression of markers at the early stages and to reduced expression markers at the late stages of myogenesis. Therefore, mutations in nuclear lamins can influence the process of muscle differentiation.

[Nuclear lamins regulate osteogenic differentiation of mesenchymal stem cells].

Nuclear lamins are the major proteins of nuclear envelope and provide the strength of nuclear membrane as well as the interaction of extra-nuclear structures with components of cell nucleus. Recently, it became clear that lamins not only play a structural role in the cell, but could also regulate cell fate, for example lamins could influence cell differentiation via interaction with components of the Notch signaling pathway. Human mutations in LMNA, encoding lamin A/C lead to diseases commonly referred to as laminopathies. Different mutations cause tissue specific phenotypes that affect predominantly a tissue of mesenchymal origin. The nature of this phenomenon, as well as the mechanisms by which lamins regulate cell differentiation remain poorly understood. The aim of this study was to investigate the effect of different mutations of the LMNA on human mesenchymal stem cell (MSC) osteogenic differentiation, and to explore a possible interaction of lamins and Notch signaling pathway. We modified human MSC with mutant LMNA bearing known mutations with tissue specific phenotype associated with different laminopathies. We have shown that mutations associated with different diseases have different effects on the efficiency of MSC osteogenic differentiation and on the expression of specific osteogenic markers SPP1, IBSP and BGLAP. We have also shown that one of the mechanisms involved in the regulation of MSC differentiation may be an interaction of lamins A/C with components of Notch signaling.

[Lamin A/C mutations change differentiation potential of mesenchymal stem cells].

Mutations in lamin A/C gene (LMNA) lead to development of severe disorders--laminopathies. Unlike most other types of intermediate filaments, where the pathological effect of mutations is tightly linked to alteration of mechanical and integrative functions, the detailed mechanism of lamin mutations is still unclear and possibly involves the alteration of nuclear signaling and transcriptional processes. Since the mesenchymal lineage tissues such as myocardium, skeletal muscle, adipose and bone tissues are mostly affected in laminopathies, the role of lamin A/C in differentiation process of mesenchymal stem cells has been assumed. The aim of the study was to estimate the effect of LMNA mutations of differentiation of mesenchymal stem cells into adipose lineages. In vitro mitagenesis was performed on wild type LMNA gene incorporated in a lentiviral vector. Several previously described mutations in LMNA were used, each associated with a certain phenotype. Adipose-derived mesenchymal stem cells from healthy donors were transduced with lentiviruses bearing either wild-type or mutant LMNA. Cells were then induced to adipose differentiation. We show that mutant LMNA/C promotes differentiation capacity of mesenchymal stem cells as seen by morphological changes and by expression of specific adipose markers.