Essential roles of KLF4 in the homeostasis and plasticity of mesenchymal stem cells

Abstract

The best known function of Krüppel-like Factor 4 (KLF4) is perhaps being one of the four Yamanaka reprogramming factors in the generation of induced pluripotent stem cells (iPSCs). At the early stage of reprogramming, KLF4 was shown to be important in the initiation of mesenchymal-epithelial transition (MET). The induction of epithelial genes by KLF4 is consistent with its function in the epithelial tissue development and homeostasis, where it is required during the establishment of skin barrier function and intestinal tissue regeneration after injury. A role of KLF4 as a cell cycle regulator has been well established, although it is observed to be a tumor suppressor in some cancer cell lines while an oncogene in others, indicating that its function in cell cycle regulation is in a context-dependent manner. Expression of KLF4 is enriched in epithelial tissues and most studies are performed in epithelial cell lines, but increasing evidences indicate that KLF4 is also a critical regulator in diverse types of cells. In particular, studies in vascular smooth muscle cells (VSMCs) have demonstrated that KLF4 is a key modulator in VSMCs phenotype transition during vascular disease progression and regeneration after injury, supporting a regulatory role of KLF4 in mesenchymal cell biology which has not been well studied. Here, we explored the function of KLF4 in mesenchymal cell biology using human iPSC derived mesenchymal stem cells (iPSC-MSCs), a cell line generated previously by our lab. Overexpression of KLF4 strongly suppressed cell proliferation supporting a function of KLF4 as a negative cell cycle regulator in iPSC-MSCs. Cell cycle analysis showed an increase in G1-stage population while a decrease in G2/M-stage population after cell cycle synchronization by G1-stage inhibitor L-mimosine, suggesting a block in G1-S transition by KLF4. Senescence-associated β-galactosidase (SA-β-gal) staining revealed an increase in cellular senescence after KLF4 overexpression, but expressions of many senescence-associated secretory phenotype (SASP) components, such as the pro-inflammatory factors IL1B, IL6, SERPINE1 at mRNA levels and IL8 at protein level, were suppressed. RNA-sequencing (RNA-seq) was performed on iPSC-MSCs with KLF4 overexpression or knockdown, and revealed downregulations of a large group of genes associated with cell cycle by both KLF4 overexpression and knockdown, suggesting that fine-tuned expression of KLF4 is important to maintain cellular homeostasis in iPSC-MSCs. Consistent with its roles in epithelial differentiation and skin barrier function, a set of epithelial-specific genes were significantly induced by KLF4 overexpression. In addition, a subset of TGF-βs signaling genes and YAP1/TAZ target genes were significantly suppressed by KLF4 overexpression. Immunofluorescence staining of YAP1 confirmed a decrease in nuclear YAP1 translocation, indicating a reduction in YAP1/TAZ mediated signaling which probably contributed to cell cycle inhibition as well. Moreover, we observed a phenotype transition in iPSC-MSCs treated with BMP4 and retinoic acid (RA) which was fully blocked by KLF4 overexpression, supporting the function of KLF4 as a potential regulator in mesenchymal cell phenotype transition.