The diverse role of laminin isoforms in neuronal cells, human mast cells and blood platelets

Abstract: The close interaction of cells with other cells and with extracellular matrix (ECM) components is a prerequisite in a number of biological activities. During development of the nervous system, axons often navigate a considerable distance depending on the type of molecular factors they encounter. Similarly, precursor and mature cells of haematopoietic origin home to specific locations during inflammation, wound healing, immune responses and thrombosis. Laminins (LMs), major components of specialized ECMs known as basement membranes, are large heterotrimeric glycoproteins made of alphabetagamma subunits. To date, 5alpha, 3beta, and 3gamma chains have been identified, which by combination yield at least 15 different LM isoforms. These isoforms have a cell-and tissue-specific expression and are recognized by several integrins. The functional role of laminin isoforms has not been fully explored as the majority of early studies focused on LM-111 (alpha1beta1gamma1, laminin-1), the first laminin to be identified. The work presented in this thesis was therefore designed primarily to address synthesis, expression and, particularly, function of laminin isoforms in different cell types. First, we showed synthesis and expression of LM-211 (alpha2beta1gamma1, laminin-2) and LM-411 (alpha4beta1gamma1, laminin-8) w by tooth pulp fibroblasts based on RT-PCR, FACS, immunoprecipitation and Western blot analysis. In functional studies, LM-411, unlike LM-211, was shown to strongly promote neurite outgrowth from sensory trigeminal ganglion (TG) neurons. This activity may be relevant to tooth innervation. In a following study, strong adhesion and migration promoting activities of alpha3-and alpha5-LMs, but not of alpha1-, alpha2- and alpha4-LMs, for human mast cells (CBMCs and HMC.1) were demonstrated. Among the different laminin-binding integrins, alpha3beta1 mediated the cell adhesion and migration. HMC-1 cells expressed transcripts for LM alpha5, beta1 and gamma1 and, following stimulation, secreted the corresponding heterotrimer LM-511 (alpha5beta1gamma1). These findings demonstrated the pivotal role of alpha3- and alpha5-LMs and their alpha3beta1 integrin receptor in mast cell adhesion and migration, and may explain the characteristic tissue localization of these immune cells in close apposition to epithelial, vascular and neural basement membranes. Synthesis of laminins by erythromegakaryocytic cell lines and their secretion by blood platelets were also investigated. Other than detecting transcripts for LM alpha3, alpha5, beta1, beta2, and gamma1 in the cell lines, presence of fully heterotrimeric alpha-3 (LM-311 and LM-321) and alpha-5 (LM-511 and LM-521) laminins was demonstrated in these cells as well as in platelets. Both alpha3- and alpha5-LMs were secreted by the platelets following stimulation. Functional studies showed that LM-511 (Lm-10) was the most platelet adhesive isoform followed by LM-411 (LM-8), LM-332 (LM-5) and LM-111 (LM-1). This adhesion was largely mediated by alpha6beta1 integrin. In spite of their adhesive properties, LM-332, LM-411 and LM-511 induced neither P-selectin expression nor cell aggregation, two signs of platelet activation. In addition, we reported expression of alpha3-LMs in blood vessels of skin, gingiva, lymph nodes and other tissues, mainly as LM-311 and/or LM-321. Moreover, migration promoting and platelet-like particle forming activities of major vascular laminin isoforms LM-411 and LM-511 were demonstrated on in vitro differentiated CD41+ megakaryocytes, in comparison to other laminin isoforms. Thus, vascular laminins may contribute, when exposed to the circulation, to platelet adhesion but not activation and, in bone marrow, to megakaryocyte migration and platelet formation. Altogether, this thesis work illustrates the diverse functional role of laminin isoforms in different cell types.