Never in mitosis gene A-related kinase-6 deficiency deteriorates diabetic cardiomyopathy via regulating heat shock protein 72.

NIMA (never in mitosis, gene A)-related kinase-6 (NEK6), a cell cycle regulatory gene, was found to regulate cardiac hypertrophy. However, its role in diabetes-induced cardiomyopathy has not been fully elucidated. This research was designed to illustrate the effect of NEK6 involved in diabetic cardiomyopathy. Here we used a streptozotocin (STZ)-induced mice diabetic cardiomyopathy model and NEK6 knockout mice to explore the role and mechanism of NEK6 in diabetic-induced cardiomyopathy. NEK6 knockout mice and wild-type littermates were subjected to STZ injection (50 mg/kg/day for 5 days) to induce a diabetic cardiomyopathy model. As a result, 4 months after final STZ injection, DCM mice revealed cardiac hypertrophy, fibrosis, and systolic and diastolic dysfunction. NEK6 deficiency causes deteriorated cardiac hypertrophy, fibrosis, and cardiac dysfunction. Furthermore, we observed inflammation and oxidative stress in the hearts of NEK6 deficiency mice under diabetic cardiomyopathy pathology. Adenovirus was used to upregulate NEK6 in neonatal rat cardiomyocytes, and it was found that NEK6 ameliorated high glucose-induced inflammation and oxidative stress. Our findings revealed that NEK6 increased the phosphorylation of heat shock protein 72 (HSP72) and increased the protein level of PGC-1α and NRF2. Co-IP assay experiment confirmed that NEK6 interacted with HSP72. When HSP72 was silenced, the anti-inflammation and anti-oxidative stress effects of NEK6 were blurred. In summary, NEK6 may protect diabetic-induced cardiomyopathy by interacting with HSP72 and promoting the HSP72/PGC-1α/NRF2 signaling. KEY MESSAGES: NEK6 knockout deteriorated cardiac dysfunction, cardiac hypertrophy, fibrosis as well as inflammation response, and oxidative stress. NEK6 overexpression attenuated high glucose induced inflammation and oxidative stress. The underlying mechanisms of the protective role of NEK6 in the development of diabetic cardiomyopathy seem to involve the regulation of HSP72-NRF2- PGC-1α pathway. NEK6 may become a new therapeutic target for diabetic cardiomyopathy.

Role of Exogenous Hsp72 on Liver Dysfunction during Sepsis.

This study examined the role of exogenous heat shock protein 72 (Hsp72) in reversing sepsis-induced liver dysfunction. Sepsis was induced by cecal ligation and puncture. Liver function was determined on the basis of the enzymatic activities of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). Apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP) protein expressions were analyzed using Western blotting. Results showed GOT and GPT levels increased during sepsis, and levels were restored following the administration of human recombinant Hsp72 (rhHsp72). Increased liver tissue apoptosis was observed during sepsis, and normal apoptosis resumed on rhHsp72 administration. The Bcl-2/Bax ratio, cleaved caspase-3, caspase-9, and PARP protein expressions in the liver tissues were upregulated during sepsis and normalized after rhHsp72 treatment. We conclude that, during sepsis, exogenous Hsp72 restored liver dysfunction by inhibiting apoptosis via the mitochondria-initiated caspase pathway.

Treatment with recombinant Hsp72 suppresses collagen-induced arthritis in mice.

Although the level of heat shock protein (Hsp72) has been shown to be enhanced in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid, it remains unclear what role extracellular Hsp72 plays in the pathogenesis of RA. This study was conducted to investigate the effects of recombinant human Hsp72 on collagen-induced arthritis (CIA) when administered therapeutically and elucidate its underlying mechanism. We demonstrated that recombinant Hsp72 significantly reduced disease severity. Hsp72-treated animals displayed significantly less cartilage and bone destruction than that in the controls. Hsp72 treatment also reduced the expression of tumor necrosis factor alpha and interleukin 6 in the sera. Furthermore, Hsp72 treatment significantly inhibited activation of nuclear factor kappa B (NF-κB) in synovial tissues of CIA mice. These findings suggest that recombinant Hsp72 effectively suppressed synovial inflammation and the development and progress of CIA, which is mediated through the reduction of production of proinflammatory cytokines and the suppression of activation of NF-κB pathway.

Chaperone-rich cell lysate embedded with BCR-ABL peptide demonstrates enhanced anti-tumor activity against a murine BCR-ABL positive leukemia.

Chaperone proteins are effective antitumor vaccines when purified from a tumor source, some of which are in clinical trials. Such vaccines culminate in tumor-specific T cell responses, implicating the role of adaptive immunity. We have developed a rapid and efficient procedure utilizing an isoelectric focusing technique to obtain vaccines from tumor or normal tissues called chaperone-rich cell lysate (CRCL). Tumor-associated peptides, the currency of T cell-mediated anticancer immunity, are believed to be purveyed by chaperone vaccines. Our purpose was to demonstrate our ability to manipulate the peptide antigen repertoire of CRCL vaccines as a novel anticancer strategy. Our methods allow us to prepare "designer" CRCL, utilizing the immunostimulation activity and the carrying capacity of CRCL to quantitatively acquire and deliver exogenous antigenic peptides (e.g., derived from the oncogenic BCR/ABL protein in chronic myelogenous leukemia). Using fluorescence-based and antigen-presentation assays, we determined that significant quantities of exogenously added peptide could accumulate in "designer" CRCL and could stimulate T cell activation. Further, we concluded that peptide-embedded CRCL, devoid of other antigens, could generate potent immunity against pre-established murine leukemia. Designer CRCL allows for the development of personalized vaccines against cancers expressing known antigens, by embedding antigens into CRCL derived from normal tissue.