Splice Wars: The Role of MLCK Isoforms in Ventilation-induced Lung Injury

Acute respiratory distress syndrome (ARDS) is caused primarily by inflammatory injury to the alveolar epithelial-endothelial barrier, which leads to the accumulation of protein-rich pulmonary edema, severe hypoxemia, and impaired carbon dioxide excretion (1, 2). Despite the high mortality rate associated with ARDS (25–35%), no therapeutic options exist outside of supportive care with mechanical ventilation (1, 2). However, mechanical ventilation may be a double-edged sword: it may be indispensable to preserve life, but it can also lead to capillary leakage and acute lung inflammation, thereby exacerbating or causing lung injury through a variety of mechanisms referred to as ventilator-induced lung injury (VILI) (3, 4). Repair of the damaged endothelial barrier, a functional alveolar epithelium, and resolution of inflammation are required for recovery from lung injury. Therefore, a better understanding of the signaling pathways involved in the worsening and restoring of barrier function in response to mechanical stress is necessary for the development of effective treatments.

Previous studies have outlined the importance of endothelial cell (EC) signaling in the pathogenesis of VILI (5, 6). During mechanical ventilation at high tidal volumes, the increased stretch causes pathologic signaling, altering the gene expression profiles in both human lung EC lines and animal models of VILI (5). In an effort to reduce the occurrence of VILI, clinical guidelines are being adjusted to include protective lung ventilation strategies, such as the use of low tidal volumes (6 ml/kg of predicted body weight) together with increased positive end-expiratory pressure (PEEP). However, therapeutic options for specific treatment of endothelial permeability disorders in VILI have yet to be developed (7). Mechanical stress is signaled through a complex network that includes cell-to-cell junction proteins (e.g., adherens junctions, tight junctions, and focal adhesions) and can activate several signaling pathways, including ion channels, G proteins, and protein kinases (6), with subsequent phosphorylation of cytoskeletal proteins such as nonmuscle myosin light-chain kinase (nmMLCK) (8). Because of its ability to regulate actomyosin contractility in ECs stimulated with agonists such as thrombin, nmMLCK has been described as an essential determinant of endothelial barrier integrity (2). Among the several isoforms of nmMLCK identified thus far, nmMLCK2 is the predominant isoform in the lung endothelium and lacks exon 11 due to alternative splicing (8, 9). As a consequence, nmMLCK2 lacks the canonical regulatory phosphorylation sites (Y464, Y471) present in nmMLCK1 which are involved in the translocation to the actin cytoskeleton and promote the restoration of the barrier (9). Consistent with this, studies using mice overexpressing nmMLCK2 revealed worsened VILI, suggesting that a switch from nmMLCK1, which functions in barrier restoration, to nmMLCK2 causes a more inflammatory environment (10). Recently, SNPs in MYLK, the gene coding for nmMLCK, have been reported to be associated with ARDS risk and mortality, suggesting that nmMLCK plays a role in human disease (11). Together, these published reports demonstrate the importance of this pathologic isoform in the progression of lung injury.

Pre-mRNA splicing takes place in a macromolecular ribonucleoprotein assembly called the spliceosome. A component of the spliceosome, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is an RNA-binding protein that is involved in processing heterogeneous nuclear RNA into mRNA (12). Two isoforms of hnRNPA1 have been well characterized: a larger, highly active 1-A isoform and a smaller but more predominant 1-B isoform with reduced activity (12, 13). A variety of sequence motifs have been described for hnRNPA1, with posttranslational modifications potentially regulating the selection of targets (12). As hnRNPA1 is both highly conserved and abundantly expressed, investigators are continuing to identify new targets of the splicing factor.

In this issue of the Journal, Mascarenhas and colleagues (pp. 604–613) provide compelling evidence that hnRNPA1 regulates the expression of proinflammatory nmMLCK2 (14). In their study, the authors demonstrate the functional relevance of insights gained from bioinformatics in silico approaches, showing a direct interaction of hnRNPA1 with intron 10–11 of the MYLK gene, and upregulation of hnRNPA1 in lung samples from mice subjected to VILI, as well as cultured human ECs exposed to cyclic stretch (CS). Using a mini-gene approach, i.e., transfecting cells with a construct that expresses only the gene segment of interest (exon 10–12) (15), the authors show that increases in exon skipping, and thus increased alternative splicing of nmMLCK2, correlate with increased expression of hnRNAP1. They also used the mini-gene construct to overexpress wild-type MYLK and MYLK with the putative hnRNPA1 binding site mutated. Overexpression of the mutated construct resulted in a loss of the MYLK–hnRNPA interaction and an increase in the nonpathogenic isoform of nmMLCK, nmMLCK1, simultaneously with a decrease in nmMLCK2. Importantly, the authors found that reductions in hnRNPA1 levels prevented the CS-induced increase in nmMLCK2 expression; however, the levels of nmMLCK remained as high as in cells treated with a control siRNA and exposed to CS. This discrepancy could be due to insufficient silencing combined with a redundancy in the expressed hnRNPA1-A/B isoforms, as the authors found that both hnRNPA isoforms were capable of interacting with nmMLCK.

The authors used a multifactorial approach to validate hnRNPA1 as a key component in regulating susceptibility to the vascular barrier dysfunction that drives VILI; however, their study has some limitations. Their quantification of expression during experimental VILI was performed in whole lung, obscuring the true expression profile in the specific cell type of interest. The experimental evidence would have been strengthened by isolation of a purified population of primary murine ECs in an experimental model of VILI. They performed the majority of their mechanistic experiments using overexpression of constructs, creating an artificial system to study protein–protein interactions. With regard to the physiological interaction between hnRNPA1 and nmMLCK, immunoprecipitation of endogenous protein complexes could reveal more relevant preferences for nmMLCK binding to the different isoforms of hnRNPA1. Another discordant fact is that even though the authors have previously described that silencing of nmMLCK2 enhances barrier responses in ECs (15), silencing of hnRNPA1 in lung ECs does not protect the cells from a thrombin-induced increase in permeability. Future studies may be needed to resolve this discrepancy.

The pulmonary endothelium orchestrates the accumulation of fluid and the consequent inflammatory milieu that develops during ARDS and VILI. The study by Mascarenhas and colleagues provides a new therapeutic target in vascular barrier dysregulation, as expression of hnNRPA1 due to increased amounts of the proinflammatory isoform nmMLCK2 may contribute to the risk and severity of ARDS/VILI.