JAK2 Inhibition by Fedratinib Reduces Osteoblast Differentiation and Mineralisation of Human Mesenchymal Stem Cells

Nihal AlMuraikhi,Amal Alotaibi,Musaad Alfayez,Hanouf Alaskar,Moustapha Kassem,Sarah Binhamdan

doi:10.3390/molecules26030606

Abstract

Several signalling pathways, including the JAK/STAT signalling pathway, have been identified to regulate the differentiation of human bone marrow skeletal (mesenchymal) stem cells (hBMSCs) into bone-forming osteoblasts. Members of the JAK family mediate the intracellular signalling of various of cytokines and growth factors, leading to the regulation of cell proliferation and differentiation into bone-forming osteoblastic cells. Inhibition of JAK2 leads to decoupling of its downstream mediator, STAT3, and the subsequent inhibition of JAK/STAT signalling. However, the crucial role of JAK2 in hBMSCs biology has not been studied in detail. A JAK2 inhibitor, Fedratinib, was identified during a chemical biology screen of a small molecule library for effects on the osteoblastic differentiation of hMSC-TERT cells. Alkaline phosphatase activity and staining assays were conducted as indicators of osteoblastic differentiation, while Alizarin red staining was used as an indicator of in vitro mineralised matrix formation. Changes in gene expression were assessed using quantitative real-time polymerase chain reaction. Fedratinib exerted significant inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells, as demonstrated by reduced ALP activity, in vitro mineralised matrix formation and downregulation of osteoblast-related gene expression, including ALP, ON, OC, RUNX2, OPN, and COL1A1. To identify the underlying molecular mechanisms, we examined the effects of Fedratinib on a molecular signature of several target genes known to affect hMSC-TERT differentiation into osteoblasts. Fedratinib inhibited the expression of LIF, SOCS3, RRAD, NOTCH3, TNF, COMP, THBS2, and IL6, which are associated with various signalling pathways, including TGFβ signalling, insulin signalling, focal adhesion, Notch Signalling, IL-6 signalling, endochondral ossification, TNF-α, and cytokines and inflammatory response. We identified a JAK2 inhibitor (Fedratinib) as a powerful inhibitor of the osteoblastic differentiation of hMSC-TERT cells, which may be useful as a therapeutic option for treating conditions associated with ectopic bone formation or osteosclerotic metastases.

Highlights

Janus kinase (JAK)/signalling transducers and activators of transcription (STAT)signalling has been identified as the downstream signalling of a wide array of growth factors, hormones, and cytokines that regulate different cellular processes, including cell proliferation, differentiation, cellular senescence, and apoptosis [1,2,3,4,5,6,7,8]
We reported the results of a small molecule library screen that recognised various small molecule inhibitors with diverse effects on the osteoblastic differentiation of hMSC-TERT cells using Alkaline phosphatase (ALP) activity quantification as a readout [15]
We first studied the effect of continuous exposure to in vitro treatment of hMSC-TERT with Fedratinib at increasing concentrations

Summary

Introduction

Janus kinase (JAK)/signalling transducers and activators of transcription (STAT)signalling has been identified as the downstream signalling of a wide array of growth factors, hormones, and cytokines (such as interferon-γ and interleukin-6 family members) that regulate different cellular processes, including cell proliferation, differentiation, cellular senescence, and apoptosis [1,2,3,4,5,6,7,8]. The JAK family is composed of four members, JAK1, JAK2, JAK3 and the tyrosine-protein kinase 2 (Tyk). The JAK family is composed of four members, JAK1, JAK2, JAK3 and the tyrosine-protein kinase 2 (Tyk2) These proteins are widely expressed, with the exception of JAK3, which is highly expressed in leukocytes and haematopoietic cells [1,3,4,6,7]. The members of this family have a unique structure of four domains, i.e., the FERM, SH2, pseudokinase, and kinase domains [4,8,9]. The STAT family has seven members, STAT1, STAT2, STAT3, STAT4, STAT5α, STAT5β, and STAT6, that contain SH2 functional domains to enable binding to tyrosine residues and

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Conclusion