HBX-41108

Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with self-renewal capabilities and multilineage differentiation potential, including osteogenesis. Although protein deubiquitinases have been linked to stem cell fate determination, whether protein deubiquitination contributes to lineage commitment during osteogenic differentiation of hASCs remains to be investigated. The objective of this study was to evaluate the effects of the ubiquitin specific protease 7 (USP7) on osteogenic differentiation of hASCs.

An osteocalcin promoter driven luciferase reporter system was established to initially discover the potential association between USP7 and hASC osteogenesis. To further characterize the function of USP7 in osteogenic differentiation of hASCs, a combination of in vitro and in vivo experiments were carried out through genetic depletion or overexpression of USP7 using a lentiviral strategy. Moreover, HBX 41,108, a cyanoindenopyrazine-derived deubiquitinase inhibitor of USP7, was utilized at different doses to further examine whether USP7 regulated osteogenic differentiation of hASCs through its enzymatic activity.

We demonstrated that USP7 depletion was associated with remarkable downregulation of the reporter gene activity. Genetic depletion of USP7 by lentiviral RNAi markedly suppressed hASC osteogenesis both in vitro and in vivo, while overexpression of USP7 enhanced the osteogenic differentiation of hASCs. Notably, chemical blockade via the small molecular inhibitor HBX 41,108 could efficiently mimic the effects of USP7 genetic depletion in a dose-dependent manner.

Taken together, our study revealed that protein deubiquitinase USP7 is an essential player in osteogenic differentiation of hASCs through its catalytic activity, and supported the pursuit of USP7 as a potential target for modulation of hASC-based stem cell therapy and bone tissue engineering.

Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 neuron‐restrictive silencer element (NRSE) functions as a negative regulator dependent on neuron‐restrictive silencer factor (NRSF) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE‐mediated repression of the human TPH2 (hTPH2) promoter activity in RN46A cells, a cell line derived from rat raphe neurons. Quantitative real‐time RT‐PCR analysis revealed the expression of serotonergic marker genes (Mash1, Nkx2.2, Gata2, Gata3, Lmx1b, Pet‐1, 5‐Htt, and Vmat2) and Nrsf gene in RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF‐dependent repression of the hTPH2 promoter activity. The hTPH2 promoter activity was increased by knockdown of NRSF, or over‐expression of the engineered NRSF (a dominant‐negative mutant or a DNA‐binding domain and activation domain fusion protein). MS‐275, a class I histone deacetylase (HDAC) inhibitor, was found to be more potent than MC‐1568, a class II HDAC inhibitor, in enhancing the hTPH2 promoter activity. Furthermore, treatment with the ubiquitin‐specific protease 7 deubiquitinase inhibitors, P‐22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE‐mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin‐specific protease 7‐mediated deubiquitination and stabilization of NRSF.image