14-3-3β paralog is inhibited by acetylation during differentiation to the osteogenic lineage.

Abstract

Background 14-3-3 protein family binds and regulate hundreds of serine/threonine phosphorylated proteins. Considered as redundant, ubiquitous and constantly expressed, this protein family was treated as an accessory for many signaling systems. Recently, specific biological functions were discovered for each of its seven paralogs in humans. Here, we studied the reversible inhibition by acetylation of its essential N- ε -lysine 49/51 residue during the osteogenic differentiation of human adipose-derived stem cells. Methods Human adipose-derived stem cells were obtained from healthy patients who underwent a dermolipectomy. The material was processed to obtain the ASCs from the stromal vascular fraction. Proteins of interest were analyzed by confocal microscopy and Western blot, and the expression level of selected proteins were modified by using lentivirus and specific shRNAs. Results We found that during the differentiation of ASC, the levels of 14-3-3 acK49/51 increased, showing that inhibition of 14-3-3 is necessary during this process. Among the 7 paralogs of this family, the inhibition by this posttranslational modification occurs mostly on the paralog β , located specifically in the nucleus, where 14-3-3 was described to bind to H3 histone and many transcription factors. Paralog specific inhibition by short hairpin RNA showed that silencing of 14-3-3 β gene, but not 14-3-3γ, increases significantly the osteogenic potential of the cells. Transdifferentiation from osteogenic to adipogenic linage of cell lines that were specifically silenced for 14-3-3 β o γ paralogs showed that 14-3-3b could be a negative regulator of the differentiation to the osteogenic lineage. Conclusion We showed that specifically 14-3-3 β is a negative regulator of osteogenesis. The levels of its inhibition by acetylation on lysine 51 is the key cellular mechanism to regulate it. The HBO1 acetyltransferase could be the enzyme responsible of this modification. Cells were this inhibition was emulated by decreasing the levels of expression of 14-3-3 β showed significant increased potential for the differentiation to the osteogenic lineage.