Osteoclasts on attachment to bone surface or
Osteoclasts on attachment to bone surface or culture vessel reorganize their Nifurtimox cytoskeleton to form a sealing zone, wherein they create an acidic environment and secrete bone resorbing enzymes. They also form integrin-based and F-actin rich unique cell adhesion structures called podosomes which are crucial for cell adhesion, spreading, migration, and thus for bone resorption . Estrogen disrupted the actin ring formation and also decreased the podosome density in osteoclasts transfected with non-targeting siRNA. Moreover, these osteoclasts displayed defective polarization. Kim et al reported that LYN−/− osteoclast culture has more number of cells with defined actin ring upon stimulation with elevated levels of RANKL . They also report an increase in the number of resorption pits formed by these osteoclasts on dentine slices. Corroborative to their findings, we also observed LYN knockdown osteoclasts with proper actin ring formation and resorbed larger area on dentine slices. Interestingly, LYN knockdown osteoclasts differentiated in presence of estrogen showed defined actin ring formation with significantly high podosome density and showed no impaired polarization and no reduction in area resorbed on dentine slices as compared to osteoclasts transfected with non-targeting siRNA differentiated in presence of estrogen. This suggests that LYN knockdown has a pro-osteoclastogenic effect and protects osteoclasts from the inhibitory effect of estrogen.
To summarize, we show that expression and activity of LYN, a negative regulator of osteoclastogenesis, is upregulated by estrogen and is required for an estrogen-dependent phenomena which includes decelerating osteoclastogenesis and reducing bone resorption potential of osteoclasts. Further, we have conclusively elucidated that LYN is required for estrogen-dependent effective inhibition of the activation of NFATc1 and c-Src for suppression of calcium signaling during osteoclastogenesis. In addition, we showed that in LYN knockdown osteoclasts, estrogen failed to induce apoptosis and also reduce the production of bone resorbing enzymes. Moreover, these cells were protected from disruption of sealing zone formation and showed high podosome density, enabling them to migrate, spread and attach more efficiently on the bone surface. These osteoclasts resorbed abundant area on dentine slices. Thus, this study provides the first line of evidence of the role of LYN as a significant signaling mediator of the inhibitory effect of estrogen on human osteoclast differentiation, survival, and function. Future in vivo studies are required to analyze the effect of estrogen on bone mass in LYN knockout animal models which may produce corroborative findings to support and broaden the understanding of this link. Consequently, it may pave the path towards the development of novel therapeutic strategies for increasing the expression and/or activation of LYN during osteoclastogenesis for improved management of bone resorptive pathology.
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Conflicts of interest
Acknowledgments The authors are thankful to Dr. Smita D. Mahale, Director of NIRRH, and Dr. S. D. Kholkute, Ex-Director of NIRRH, for encouraging and supporting the work. The study was supported by Indian Council of Medical Research (Accession no: RA/669/08-2018) and Department of Science and Technology (DST, Project no: EMR/2015/001595/HS), Government of India. Authors acknowledge University Grants Commission and DST for awarding fellowships to SG. The contribution of Dr. Sandip Chavan during initial sample processing for iTRAQ labeling is also acknowledged. The authors thank the team of the confocal facility (Dr. Nafisa Balasinor, Mrs. Reshma Gaonkar, and Mrs. Sobha Sonavane) and flow cytometry facility (Dr. Srabani Mukherjee and Mrs. Gayatri Shinde) for providing the assistance with indirect immunofluorescence and flow cytometry respectively. The authors thank Ms. Seema Kadam for providing the necessary technical assistance.