Pleiotropic effects of extracellular vesicles from induced pluripotent stem cell-derived cardiomyocytes on ischemic cardiomyopathy: A preclinical study.

BACKGROUND: Stem cell-secreted extracellular vesicles (EVs) play essential roles in intercellular communication and restore cardiac function in animal models of ischemic heart disease. However, few studies have used EVs derived from clinical-grade stem cells and their derivatives with stable quality. Moreover, there is little information on the mechanism and time course of the multifactorial effect of EV therapy from the acute to the chronic phase, the affected cells, and whether the effects are direct or indirect. METHODS: Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) were produced using a clinical-grade differentiation induction system. EVs were isolated from the conditioned medium by ultracentrifugation and characterized in silico, in vitro, and in vivo. A rat model of myocardial infarction was established by left anterior descending artery ligation and treated with iPSCM-derived EVs. RESULTS: iPSCM-derived EVs contained microRNAs and proteins associated with angiogenesis, antifibrosis, promotion of M2 macrophage polarization, cell proliferation, and antiapoptosis. iPSCM-derived EV treatment improved left ventricular function and reduced mortality in the rat model by improving vascularization and suppressing fibrosis and chronic inflammation in the heart. EVs were uptaken by cardiomyocytes, endothelial cells, fibroblasts, and macrophages in the cardiac tissues. The pleiotropic effects occurred due to the direct effects of microRNAs and proteins encapsulated in EVs and indirect paracrine effects on M2 macrophages. CONCLUSIONS: Clinical-grade iPSCM-derived EVs improve cardiac function by regulating various genes and pathways in various cell types and may have clinical potential for treating ischemic heart disease.

LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells.

The epithelial-to-mesenchymal transition (EMT) is a critical process by which cancer cells acquire malignant features. However, the molecular mechanism and functional implications of EMT and the mesenchymal-to-epithelial transition (MET) in tumor progression remain elusive. In this study, we established two aggressive cancer cell lines from the human oral cancer cell line SAS, mesenchymal-like SAS-m4 and epithelial-like SAS-δ. SAS-δ is a revertant cell obtained by inducing MET in SAS-m4. SAS-δ, but not SAS-m4, exhibited abnormal cell growth, including piled-up overgrowth and invasive tumor formation in the tongues of nude mice, suggesting that SAS-δ represented more advanced cancer cells than the parental SAS cells. EMT-related transcriptional factor SLUG is phosphorylated at T208 and partly stabilized by the Hippo pathway kinases, LATS1 and LATS2. Depletion of SLUG promoted the invasive activity of SAS-δ by increasing the protein levels of LATS1/2 and the proportion of the phosphorylated form among total SLUG protein. Our results suggest that the LATS1/2-SLUG axis regulates the transition of SAS cells to the advanced stage via repeated switching between EMT and MET. Therefore, an anti-SLUG-pT208 antibody would be valuable not alone as a malignant tumor marker antibody but also as a prognostic tool for patients with malignant disease.

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B'γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan-Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis.

The tumor suppressor kinases LATS1 and LATS2 (LATS1/2) regulate not only organ size through the Hippo signaling pathway, but also cell-cycle checkpoints and apoptosis via other signaling cascades. We previously reported that LATS1/2 localize to the mitotic apparatus, where they are involved in the phosphorylation and activation of the mitotic kinase Aurora-B; however, the detailed mechanism of LATS1/2 action remains obscure. The activity of Aurora-B is stringently regulated by formation of the chromosomal passenger complex containing the inner centromere protein (INCENP), which leads to appropriate activation of Aurora-B during mitosis and cytokinesis. In this study, we found that LATS1/2 phosphorylated INCENP at S894 in the Thr-Ser-Ser motif. Moreover, the LATS-mediated phosphorylation of S894 was necessary and sufficient for the activation of Aurora-B, which is required for completion of cytokinesis in cells engaged in multipolar division. We propose a novel mechanism for regulation of Aurora-B via INCENP phosphorylation by LATS1/2 during cytokinesis.

LATS2 Positively Regulates Polycomb Repressive Complex 2.

LATS2, a pivotal Ser/Thr kinase of the Hippo pathway, plays important roles in many biological processes. LATS2 also function in Hippo-independent pathway, including mitosis, DNA damage response and epithelial to mesenchymal transition. However, the physiological relevance and molecular basis of these LATS2 functions remain obscure. To understand novel functions of LATS2, we constructed a LATS2 knockout HeLa-S3 cell line using TAL-effector nuclease (TALEN). Integrated omics profiling of this cell line revealed that LATS2 knockout caused genome-wide downregulation of Polycomb repressive complex 2 (PRC2) and H3K27me3. Cell-cycle analysis revealed that downregulation of PRC2 was not due to cell cycle aberrations caused by LATS2 knockout. Not LATS1, a homolog of LATS2, but LATS2 bound PRC2 on chromatin and phosphorylated it. LATS2 positively regulates histone methyltransferase activity of PRC2 and their expression at both the mRNA and protein levels. Our findings reveal a novel signal upstream of PRC2, and provide insight into the crucial role of LATS2 in coordinating the epigenome through regulation of PRC2.

Phosphorylation of CHO1 by Lats1/2 regulates the centrosomal activation of LIMK1 during cytokinesis.

Large tumor suppressor 1 and 2 (Lats1/2) regulate centrosomal integrity, chromosome segregation and cytokinesis. As components of the centralspindlin complex, the kinesin-like protein CHO1 and its splicing variant MKLP1 colocalize with chromosome passenger proteins and GTPases and regulate the formation of the contractile ring and cytokinesis; however, the regulatory mechanisms of CHO1/MKLP1 remain elusive. Here, we show that Lats1/2 phosphorylate Ser716 in the F-actin-interacting region of CHO1, which is absent in MKLP1. Phosphorylated CHO1 localized to the centrosomes and midbody, and the actin polymerization factor LIM-kinase 1 (LIMK1) was identified as its binding partner. Overexpression of constitutively phosphorylated and non-phosphorylated CHO1 altered the mitotic localization and activation of LIMK1 at the centrosomes in HeLa cells, leading to the inhibition of cytokinesis through excessive phosphorylation of Cofilin and mislocalization of Ect2. These results suggest that Lats1/2 stringently control cytokinesis by regulating CHO1 phosphorylation and the mitotic activation of LIMK1 on centrosomes.

CAWS administration increases the expression of interferon γ and complement factors that lead to severe vasculitis in DBA/2 mice.

BACKGROUND: Candida albicans water-soluble fraction (CAWS), a mannoprotein-ß-glucan complex obtained from the culture supernatant of C. albicans NBRC1385, causes CAWS-mediated vasculitis (CAWS-vasculitis) in B6 and DBA/2 mice with mild and lethal symptoms, respectively. Why CAWS is lethal only in DBA/2 mice remains unknown. RESULTS: We performed DNA microarray analyses using mRNA obtained from peripheral blood mononuclear cells (PBMCs) of B6 and DBA/2 mice and compared their respective transcriptomes. We found that the mRNA levels of interferon-γ (Ifng) and several genes that regulate the complement system, such as C3, C4, Cfb, Cfh, and Fcna, were increased dramatically only in DBA/2 mice at 4 and 8 weeks after CAWS administration. The dramatic increase was confirmed by quantitative real-time polymerase chain reactions (qRT-PCR). Moreover, mRNA levels of immune-related genes, such as Irf1, Irf7, Irf9, Cebpb, Ccl4, Itgam, Icam1, and IL-12rb1, whose expression levels are known to be increased by Ifng, were also increased, but only in DBA/2 mice. By contrast, the mRNA level of Dectin-2, the critical receptor for the α-mannans of CAWS, was increased slightly and similarly in both B6 and DBA/2 mice after CAWS administration. CONCLUSIONS: Taken together, our results suggest that CAWS administration induces Dectin-2 mediated CAWS-vasculitis in both B6 and DBA/2 mice and the expression of Ifng, but only in DBA/2 mice, which led to increased expression of C3, C4, Cfb, Cfh, and Fcna and an associated increase in lethality in these mice. This model may contribute to our understanding of the pathogenesis of severe human vasculitis.

Lats2 phosphorylates p21/CDKN1A after UV irradiation and regulates apoptosis.

LATS2 (Large tumor suppressor 2), a member of the conserved AGC Ser/Thr (S/T) kinase family, is a human tumor suppressor gene. Here, we show that in response to ultraviolet radiation, Lats2 is phosphorylated by Chk1 at Ser835 (S835), which is located in the kinase domain of Lats2. This phosphorylation enhances Lats2 kinase activity. Subsequently, Lats2 phosphorylates p21 at S146. p21 (CDKN1A) is a cyclin-dependent kinase (CDK) inhibitor, which not only regulates the cell cycle by inhibition of CDK, but also inhibits apoptosis by binding to procaspase-3 in the cytoplasm. Phosphorylation by Lats2 induces degradation of p21 and promotes apoptosis. Accordingly, Lats2 overexpression induces p21 degradation, activation of caspase-3 and caspase-9, and apoptosis. These findings describe a novel Lats2-dependent mechanism for induction of cell death in response to severe DNA damage.

N-terminal truncation of Lats1 causes abnormal cell growth control and chromosomal instability.

The tumor suppressors Lats1 and Lats2 are mediators of the Hippo pathway that regulates tissue growth and proliferation. Their N-terminal non-kinase regions are distinct except for Lats conserved domains 1 and 2 (LCD1 and LCD2), which may be important for Lats1/2-specific functions. Lats1 knockout mice were generated by disrupting the N-terminal region containing LCD1 (Lats1(ΔN/ΔN)). Some Lats1(ΔN/ΔN) mice were born safely and grew normally. However, mouse embryonic fibroblasts (MEFs) from Lats1(ΔN/ΔN) mice displayed mitotic defects, centrosomal overduplication, chromosomal misalignment, multipolar spindle formation, chromosomal bridging and cytokinesis failure. They also showed anchorage-independent growth and continued cell cycles and cell growth, bypassing cell-cell contact inhibition similar to tumor cells. Lats1(ΔN/ΔN) MEFs produced tumors in nude mice after subcutaneous injection, although the tumor growth rate was much slower than that of ordinary cancer cells. Yap, a key transcriptional coactivator of the Hippo pathway, was overexpressed and stably retained in Lats1(ΔN/ΔN) MEFs in a cell density independent manner, and Lats2 mRNA expression was downregulated. In conclusion, N-terminally truncated Lats1 induced Lats2 downregulation and Yap protein accumulation, leading to chromosomal instability and tumorigenesis.