Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK-/- C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK-/- cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.

Bcl-3 promotes Wnt signaling by maintaining the acetylation of ß-catenin at lysine 49 in colorectal cancer.

Wnt/ß-catenin signaling plays a critical role in colorectal cancer (CRC) tumorigenesis and the homeostasis of colorectal cancer stem cells (CSCs), but its molecular mechanism remains unclear. B-cell lymphoma 3 (Bcl-3), a member of the IκB family, is overexpressed in CRC and promotes tumorigenicity. Here, we report a novel function of Bcl-3 in maintaining colorectal CSC homeostasis by activating Wnt/ß-catenin signaling. Silencing Bcl-3 suppresses the self-renewal capacity of colorectal CSCs and sensitizes CRC cells to chemotherapeutic drugs through a decrease in Wnt/ß-catenin signaling. Moreover, our data show that Bcl-3 is a crucial component of Wnt/ß-catenin signaling and is essential for ß-catenin transcriptional activity in CRC cells. Interestingly, Wnt3a increases the level and nuclear translocation of Bcl-3, which binds directly to ß-catenin and enhances the acetylation of ß-catenin at lysine 49 (Ac-K49-ß-catenin) and transcriptional activity. Bcl-3 depletion decreases the Ac-K49-ß-catenin level by increasing the level of histone deacetylase 1 to remove acetyl groups from ß-catenin, thus interrupting Wnt/ß-catenin activity. In CRC clinical specimens, Bcl-3 expression negatively correlates with the overall survival of CRC patients. A significantly positive correlation was found between the expression of Bcl-3 and Ac-K49-ß-catenin. Collectively, our data reveal that Bcl-3 plays a crucial role in CRC chemoresistance and colorectal CSC maintenance via its modulation of the Ac-K49-ß-catenin, which serves as a promising therapeutic target for CRC.

Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.