NO66 suppresses muscle protein synthesis by a demethylase mechanism
Authors: zhang liping , Mitch William E. 
Published: April 2020

BackgroundCatabolic conditions like chronic kidney disease (CKD) induce muscle wasting partly by suppressing muscle protein synthesis (PS) with unknown mechanisms. We have identified a CKD‐stimulated, chromatin‐modifying, nucleolar protein 66 (NO66) and show that it suppresses both ribosomal DNA transcription and muscle protein synthesis.MethodsTransgenic mice with a loxP‐flanked (“floxed”) NO66 (NO66Flox/Flox) were a gift from Dr De Crombrugghe, University of Texas MD Anderson Cancer center, Houston, TX. Mice with global deletion of NO66 mice (NO66−/−) were created by crossing transgenic, Sox2‐cre mice with NO66Flox/Flox mice. Mice with muscle‐specific NO66 KO (MCK‐NO66) were created by crossing MCK‐Cre mice with NO66Flox/Flox mice. We studied mice with CKD (subtotal nephrectomy) with BUN >80 mg/dL, similar to values in end stage renal disease (ESRD) patients. The cancer cachexia mice were created by subcutaneously injecting with isogenic, Lewis lung carcinoma (LLC) cells. To create mice in type II diabetes: Six weeks old C57/BL6 mice were fed with a high fat diet (HFD) (58% kcal from fat, Research Diets, New Brunswick, NJ, USA) for 12 weeks. The control groups were fed the regular diet (RD: 11% kcal from fat). To create mice in acute type I diabetes: we injected 12‐week‐old C57/BL6 mice intraperitoneally with 2 doses of 150 mg/kg/d streptozotocin (STZ) (Sigma‐Aldrich) in 0.1 M citrate buffer (pH 4). Control mice were injected with the citrate buffer. 24 and 36 months old of C57/BL6 mice were used as aging mice.ResultsWhile exploring mechanisms causing defective PS, in muscles of mice or patients with CKD, we found increased expression of NO66 associated with muscle mass loss. These increases were found to be stimulated by inflammation and mediated by changes in NF‐κB: NO66 expression was suppressed by a NF‐κB inhibitor. NO66 knockout from mouse muscles blocked CKD or cancer‐induced muscle loss and improved the rates of PS. RNA‐seq identified that NO66 regulates ribosomal biogenesis. Thus, in muscle cells or muscles lacking NO66, ribosomal RNA, pre‐rRNA and PS were all increased. The mechanism involves NO66 inhibition of ribosomal DNA transcription via demethylation of H3K4me3 and H3K36me3. NO66 but not demethylase‐dead mutated NO66 decreased H3K4me3 and H3K36me3 and suppressed ribosomal RNA transcription in muscle cells. In cells lacking NO66, CHIP assays uncovered enriched histone‐tri‐methylated proteins of H3K4 and H3K36 on ribosomal DNA. Finally, we found these findings also apply to other mouse models of catabolic disorders including type II diabetes, aging and cancer cachexia.ConclusionsThe activation of NFkB in catabolic conditions stimulates the expression of NO66. NO66 causes demethylation of H3K4me3 and H3K36me36 to impair ribosomal DNA transcription, which ultimately result in decreases in PS in muscle. Thus, catabolic disorders like CKD suppress PS via epigenetic mechanisms that are mediated by NO66; blocking NO66 could devise strategies for controlling muscle catabolism.Support or Funding InformationThis work was supported by the National Institutes of Health (2R01 DK037175 to W.E.M.; T32‐DK62706 to W.E.M; Pilot/Feasibility award P30‐DK079638 to L.Z.); American Diabetic Association (1‐11‐BS‐194 to L. Z.); Norman S. Coplon extramural research grant and the support from Dr. and Mrs. Harold Selzman to L. Z.