Myosin light chain kinase ( MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

Abstract

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y⁴⁶⁴ and Y⁴⁷¹ phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y⁴⁶⁴ phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y⁴⁶⁴ phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Keywords: S1P; SNP; nmMLCK; sphingosine 1-phosphate.

Fig. 1.

nmMLCK1 Y⁴⁶⁴ phosphorylation and cellular localization upon S1P challenge. Human pulmonary microvascular ECs were transfected with EGFP-nmMLCK1 constructs with WT, S147P, or 3SNP. Then ECs were challenged with S1P (1 μM) for 2 or 5 min. Immunofluorescence assays were performed to visualize nmMLCK Y⁴⁶⁴ phosphorylation (red), F-actin (blue), and transfected EGFP-nmMLCK1 (green). These representative images were selected from > 5 independent assays.

Fig. 2.

Effects of genetic variants on nmMLCK1 Y⁴⁶⁴ phosphorylation post S1P challenge. Human pulmonary microvascular ECs were transfected with EGFP-nmMLCK1 constructs with WT, S147P, or 3SNP. Then ECs were challenged with S1P (1 μM) for 2–15 min. Immunofluorescence assays were performed to visualize nmMLCK1 Y⁴⁶⁴ phosphorylation. Relative nmMLCK1 Y⁴⁶⁴ phosphorylation levels in ECs were quantified. n = 30. *P < 0.05 compared to unstimulated WT EGFP-nmMLCK1 (the first white bar). #P < 0.05 compared to WT EGFP-nmMLCK1 group at the same time point of S1P challenge (the white bar of each time point).

Fig. 3.

Effects of genetic variants on nmMLCK1 enrichment in lamellipodia post S1P challenge. Human pulmonary microvascular ECs were transfected with EGFP-nmMLCK1 constructs with WT, S147P, or 3SNP. Then ECs were challenged with S1P (1 μM) for 2–10 min. EGFP levels in lamellipodia were quantified relative to whole cell EGFP levels. n = 30. *P < 0.05 compared to unstimulated WT EGFP-nmMLCK1 (the first white bar). #P < 0.05 compared to WT EGFP-nmMLCK1 group at the same time point of S1P challenge.