Role of WNT5A isoform L(A) and isoform S(B) during osteoblast differentiation

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Dristi Bhandari (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Karen Katula

Abstract: WNT signaling has been characterized as a critical signaling cascade in many aspects of embryogenesis, cell differentiation and tissue homeostasis. Appropriate WNT signaling requires dynamic expression of WNTs along with their receptors, ensuring a proper balance between cell differentiation and proliferation. As a result, perturbation in WNT signaling by aberrant expression underlies various congenital malfunctions, cancer and other diseases. This study is focused on one family of WNT ligands called WNT5A. WNT5A plays a central role in primary axis formation and limb bud extension in development and has regulatory functions in mesenchymal cell differentiation, including bone formation. Moreover, WNT5A exhibits tumor suppressive as well as oncogenic properties in a wide range of cancers. Notably, the WNT5A gene encodes for two protein isoforms, isoform L(A) and isoform S(B). There is evidence that the isoforms are functionally distinct in cancer. However, there is a critical gap in understanding of the functional roles of the WNT5A isoforms in normal cell function. In this study, I investigated the functional contributions of the WNT5A isoforms during the process of normal osteoblast differentiation using the human fetal osteoblast cell line, hFOB1.19. The results show an increase in transcripts of both isoforms for normal differentiating osteoblasts. A trial examined for 21 days revealed that isoform L(A) and S(B) exhibit similar pattern during differentiation where both show gradual increase with progressing days. In addition, we identified an increase in expression of the WNT5A protein in hFOB1.19 cell line. The increase in isoform expression was correlated with molecular markers of osteoblast differentiation including RUNX2, osterix and osteocalcin and alkaline phosphatase (ALP) activity. Increasing the individual isoforms L(A) and S(B) in hFOB1.19 cells did not inhibit increases in RUNX2 and osteocalcin expression. Our results suggest a slight decrease in osteocalcin levels with treatment of L(A) and S(B)- conditioned medium (CM) on day 3 compared to the control. In contrast, RUNX2 was increased at day 2 and decreased at day 3 in isoform-CM treated cells. ALP activity decreased at day 7and 10 for both L(A)-CM and S(B)-CM treated cells, but only L(A)-CM showed a significant change at day 10. Next, I attempted to knockdown the individual isoforms and total WNT5A transcripts using siRNA techniques. Knockdown was achieved total WNT5A. Results show that by knocking down WNT5A, the early differentiation marker, RUNX2 decreases significantly by day 3 whereas the later differentiation marker, osteocalcin increases significantly by day 3. Taken together, these findings suggest that WNT5A isoform L(A) and S(B) appear to be involved in osteogenesis and the onset and increases of differentiation molecular markers show correlations with increases in the isoforms. However, as both WNT5A isoforms displayed similar patterns of expression during normal differentiation and after alteration of the individual isoform levels, a functional distinction between the isoforms during osteoblast differentiation cannot be asserted.

Additional Information

Language: English
Date: 2018
Isoform A, Isoform B, Osteoblast differentiation, WNT5A, WNT5A isoforms, WNT5A knockdown
Wnt genes
Wnt proteins
Osteoblasts $x Differentiation

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