Identification and characterization of two isoforms of human megakaryoblastic Leukemia-1 and their specific regulation in myofibroblast differentiation

The reversible differentiation of various precursor cell types into myofibroblasts/cancer-associated fibroblasts (CAFs) is an important physiological as well as pathological process. Recently, the Rho-actin-MKL1-SRF pathway has been shown to regulate this process. MKL1 activity has emerged as the crucial relay adjusting the status of the actin cytoskeleton and the transcription of a substantial part of SRF target genes, including smooth muscle-specific genes. To investigate the role of MKL1 in more detail, we analyzed the exact molecular structure of the human MKL1 protein, its gene architecture, and its regulation of expression. For the first time, we describe the existence of a second human MKL1 isoform, MKL1_S, that is transcribed from an alternative promoter. In contrast to the published isoform MKL1_L, expression of this novel isoform varies strongly between different cell types and tissues. Furthermore, we identified an upstream GTG translation start of MKL1_L, resulting in a long N-terminal tail that is not present in MKL1_S. Using human adipose tissue-derived stem cells (ASCs) as a differentiation model, we were able to show that only the shorter isoform MKL1_S is strongly up-regulated within the first 24 h of TGF-β-induced myofibroblast/CAF differentiation. TGF-β constitutes the major physiological trigger of the myofibroblast/CAF differentiation program. By applying other stimuli that were reported to drive differentiation of MSCs/ASCs into the smooth muscle direction, we found that induction of MKL1_S is specific for TGF-β. To assess the mechanistic role of specific MKL1_S up-regulation, we over-expressed MKL1_S and MKL1_L in HEK293 cells and analyzed target gene expression after stimulation of the Rho-actin-MKL1 pathway. We found that MKL1_S shares the majority of its target genes with MKL1_L, including α-smooth muscle actin. However, we identified several genes that were significantly more strongly induced by MKL1_S, coding for extracellular proteins, such as MMP-16 and SPOCK-3. This MKL1_S-specific activity was mediated by a functional motif in the MKL1_S-specific N-terminal sequence. We postulate that the specific up-regulation of MKL1_S in the initial phase of TGF-β-induced myofibroblast/CAF differentiation contributes to the progression to the advanced phase, which is characterized by enhanced contractility, extracellular matrix deposition and modification, and the down-regulation of cellular migration.
Moreover, using a proteomics approach to identify MKL_1 binding proteins, we identified pyruvate kinase (PK) M1/M2 as a novel interaction partner of MKL1. PKM1/2 is an enzyme that mediates the last, rate-limiting step of glycolysis, and thereby controls the channeling of pyruvate either into the highly efficient cellular respiration process or into the less efficient lactic acid fermentation. The latter was found to be strongly increased in tumor cells, a phenomenon known as Warburg effect. The novel interaction between PKM1/2 and MKL1 might reveal a so far unknown link between tumor metabolism and MKL1-mediated cellular motility or differentiation into myofibroblasts/CAFs.