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  • br Materials and methods br Results br Discussion

    2019-07-11


    Materials and methods
    Results
    Discussion Estrogen is a potent positive regulator of bone mass and its insufficiency elicits unwarranted differentiation and activity of osteoclasts, causing excessive bone resorption and thus leads to increased risk of osteoporosis observed in postmenopausal women [[5], [6], [7]]. However, the precise mechanism through which Acarbose regulates osteoclast differentiation and activity is not well understood. Towards this, we differentiated human primary monocytes into osteoclasts in the presence and absence of estrogen and identified LYN, a member of the Src family of non-receptor tyrosine kinase (SFK) to be significantly upregulated on day 1 of osteoclastogenesis in response to estrogen. To the best of our knowledge, this is the first study reporting the use of human primary monocytes for the identification of estrogen-regulated proteins simultaneously during different stages of osteoclastogenesis in a single experimental setup. LYN is expressed in many tissues where it is involved in various signaling pathways regulating differentiation, proliferation, migration, and metabolism [14,34]. Besides, it has an immense potential to influence immune cell signaling and its dysregulation is associated with many leukemia phenotypes including acute myeloid leukemia, chronic myeloid leukemia and B-cell lymphocytic leukemia [[35], [36], [37]]. It is also associated with certain autoimmune diseases such as systemic lupus erythematosus and various solid tumors including glioblastoma, prostate and certain aggressive subtypes of breast cancer [[38], [39], [40], [41]]. Therefore, currently LYN is emerging as a potential therapeutic target in many diseases. The inhibitory role of LYN in osteoclast biology is reported recently [12,13]. However, this is the first study reporting the role of LYN as an important signaling intermediary for estrogen in osteoclast biology. Yoon et al reported that LYN transcript and protein levels reduced in mouse bone marrow-derived macrophages (BMMs) 24 h after stimulation with RANKL [13]. They also observed decreased levels of activated LYN in these cells. Similar to their findings, we also observed a decrease in transcript and protein levels with a reduction in its activated levels throughout osteoclastogenesis. Interestingly, monocytes, the osteoclast precursors, cultured in presence of M-CSF showed upregulation of LYN expression and activity in response to estrogen. This shows that estrogen is able to upregulate LYN in osteoclast precursors prior to their commitment to the osteoclast lineage. Further, we found the estrogen response element in LYN promoter sequence, which matches annotation for ERα, suggesting a probable direct genomic regulation of LYN transcription by estrogen. Calcium signaling is essential for osteoclastogenesis, and NFATc1 and c-Src are key players involved in the same. Yoon et al reported increased intracellular Ca2+ concentration and expression of NFATc1 in LYN silenced mouse BMMs after incubation with M-CSF and RANKL for 2 days [13]. They also reported an increase in expression and activation of c-Src in LYN silenced BMMs. Corroborative to their findings, we also observed increased intracellular Ca2+ levels in LYN knockdown cells throughout osteoclastogenesis and increased activation of NFATc1, and increased expression and activation of c-Src. Estrogen suppressed calcium signaling during osteoclastogenesis by reducing the intracellular Ca2+ levels, expression and activation of both, NFATc1 and c-Src in cells transfected with non-targeting siRNA. However, in LYN knockdown cells, estrogen failed to significantly inhibit the activation of NFATc1 and c-Src, thus its suppressive effect on calcium signaling was hampered, suggesting that estrogen may inhibit the expression of NFATc1 and c-Src in LYN knockdown cells, but expression and activation of LYN is required for estrogen-dependent inhibition of their activation. Osteoclasts are giant multinucleated cells formed by the fusion of monocytes. Osteoclasts with a higher number of nuclei have high resorption potential and are capable to substantially increase in size [42]. These multinucleated osteoclasts can resorb larger area due to their large expanse and intense production of bone resorbing enzymes. We observed that estrogen significantly reduced the number of osteoclasts formed and the osteoclasts thus formed were significantly smaller in size with a lesser number of nuclei and cytoplasmic growth. Whereas, LYN knockdown cells not only differentiated into giant cells with more number of nuclei but also exhibited a significant increase in cytoplasmic growth, suggesting the increased fusion of monocytes during differentiation and also increased transcriptional activity of the nuclei in LYN knockdown osteoclasts. However, the inhibitory effect of estrogen on osteoclast number, size, and extent of multinuclearity was significantly inadequate in LYN knockdown osteoclasts. Further, estrogen significantly reduced the expression of TRAP, cathepsin K, and CAII, which are major bone resorbing enzymes produced by osteoclasts. In LYN knockdown osteoclasts, no reduction in expression of these enzymes was observed in presence of estrogen.