Dual Inhibition of GSK3 and JAK by BIO Suppresses Osteoblast Differentiation and Mineralization of Human Mesenchymal Cells

TL;DR

BIO, a drug that inhibits GSK3 and JAK, suppresses bone cell development in human stem cells, showing unexpected negative effects on bone regeneration.

Contribution

The study reveals that BIO's dual inhibition of GSK3 and JAK suppresses osteoblast differentiation and alters key signaling pathways.

Findings

BIO significantly reduced ALP activity and matrix mineralization in hMSCs-TERT4 cells.

Wnt and TGF-β pathway genes were downregulated following BIO treatment.

BIO suppressed osteoblast commitment and differentiation despite not affecting cell viability.

Abstract

Glycogen synthase kinase-3 (GSK3) inhibition is a commonly used approach to promote osteogenic differentiation through activation of Wnt signaling. However, 6-bromoindirubin-3′-oxime (BIO), which is commonly used for GSK3 inhibition, also targets JAK/STAT, raising the possibility of dual pathway interference during osteoblast differentiation, as both GSK3 and JAK/STAT pathways are critical regulators of osteoblastogenesis. In this study, we investigated the effect of BIO on the osteoblast differentiation of hMSCs-TERT4. While BIO had no significant effect on cell viability or apoptosis, it markedly inhibited osteoblast differentiation, as evidenced by reduced ALP activity, decreased matrix mineralization, and downregulation of osteoblast-associated markers. Microarray analysis followed by qRT-PCR validation revealed downregulation of Wnt and TGF-β pathway genes. These findings show that BIO suppresses osteoblast commitment and osteogenic differentiation, accompanied by altered Wnt- and TGF-β-related gene expression. This study provides mechanistic insight into the off-target consequences of widely used small molecules and highlights the importance of dissecting pathway-specific roles in stem cell differentiation. Understanding the interplay between GSK3 and JAK signaling is essential for optimizing pharmacological strategies in skeletal regenerative medicine. This study highlights the importance of pathway selectivity when using small molecules in stem cell-based therapies for bone regeneration.

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