Comparison of the distribution and formation of lamin A, emerin, LINC complex at the outer and inner surface of the nuclear envelope in contractile and proliferative pulmonary artery smooth muscle cells

Abstract

Smooth muscles found on the walls of hollow organs are composed of spindle-shaped cells and are known as Smooth Muscle Cells (SMCs). These cells contract involuntarily to perform their functions such as tissue oxygenation and blood pressure regulation. SMCs are able to adapt easily to environmental changes as a consequence considerable plasticity. Although, smooth muscles generally exist in quiescent and contractile states under physiological condition, in the conditions, under conditions of environmental changes such as hypertension, angiogenesis or vascular calcification, the contractile phenotype transforms into an actively proliferating, synthetic phenotype. This process is called phenotypic switching, which is a mechanism that underlies many severe diseases, including pulmonary hypertension and atherosclerosis. The phenotypic switching is the transition from a contractile to a synthetic phenotype. It is influenced by many factors including signalling pathways, biochemical compounds and proteins. To date, these factors and the characteristics of contractile and synthetic phenotype have been investigated. However, the role of nuclear membrane proteins on phenotypic switching has not been studied by researchers in much detail, compared to other factors.

Therefore, I hypothesised that nuclear envelope proteins may have an important role in phenotypic switching through interaction with particular chromatin marks in particular regions. To test this I focussed on the following nuclear envelope proteins: lamin A, emerin, SUN-2 (Sad1 And UNC84 Domain Containing 2) and nesprin (nuclear envelope spectrin repeat proteins). Lamin A and emerin have ability to interact with Barrier-to-autointegration factor (BAF) and histone 3 lysine 9 dimethylation (H3K9me2) that are in relation to the modulation of gene expression. Nesprin and SUN-2 are responsible for the communication between cytoskeleton and nucleus, which can transfer mechanical cue to lamin A and emerin affecting chromatin interaction. My aim was to determine whether these proteins in some way contribute to those signalling pathways that modulate gene expression during the switch in XV phenotype, by modulating chromatin organisation. To test my hypothesis, I investigated the distribution and levels of these selected nuclear envelope proteins in acutely isolated pulmonary arterial myocytes and compare them to pulmonary arterial smooth muscle cells that had been cultured in a pro-proliferative medium for 7 days, which acted as a model for synthetic phenotype. The distribution of the nuclear membrane proteins detected in these two smooth muscle phenotypes was completely different. Moreover, the surface structures created by labelling these proteins were both diverse and phenotype-specific. In the contractile phenotype, for example, lamin covered the entire surface of the inner nuclear membrane, but also accumulated at higher density in identifiable structures, such as peripheral ridges, clefts and nuclear invaginations. As a result, although emerin formed similar structures, it was less uniform in distribution. The distribution of nesprin, on the other hand, was restricted to the periphery and SUN-2 was present at peripheral structures. Barrier-to-autointegration factor (BAF) and the histone mark H3K9me2 were found to be associated with emerin and lamin at the nuclear periphery, providing sites for chromatin attachment and gene repression. Remarkably, reconfiguration of the distribution and structural organisation of all proteins was observed in proliferating cells. For example, the periphery structures such as ridges and clefts as well as nuclear invaginations were not observed throughout the proliferating nuclei. Instead of these structures, lamin A accumulation was observed, which caused some indentations including Otype and I-type structures. As in the contractile phenotype, lamin A distribution covered the area underneath the nuclear envelope and emerin also showed similar pattern. However, the distribution of nesprin in proliferating nuclei was observed as puncta whereas SUN-2 was located on particular regions.

Dependent on these changes in the distribution of proteins, alterations in their colocalization with BAF and histone mark, H3K9me2 was detected, which is that the association of lamin A and H3K9me2 spreads through the nuclei in proliferating nuclei, instead of located on particular structures. This colocalization regions was observed as different pattern in different ridges identified. Moreover, the colocalization between lamin A and H3K9me2 reduced from contractile to proliferative phenotype. Interestingly, BAF and emerin colocalization increased in proliferating phenotype, compared to contractile phenotype and the ridge specific distribution of colocalization puncta was also observed in this proteins association.

Overall, these results indicate that this redistribution of nuclear membrane proteins may correspond to an important role in SMC phenotypic switching, through gene expression modulation by changing chromatin organisation.