| Abstract |
The osteoclast is characterized as the ‘sole’ bone-resorbing cell, and this is due to its unique characteristics, that provide osteoclast with the ability to dissolve organic and mineral bone. An important feature of the osteoclast is the fact that osteoclasts’ development and function are largely controlled by the microenvironment, especially by pro-inflammatory cytokines such as IL-1 and TNFa. Therefore, osteoclast formation and function are enhanced in inflammatory situations, such as rheumatoid arthritis, leading to the clinical feature of osteopenia and osteoporosis, a condition of systemic reduced bone mass. Finally, studying molecules associated with the differentiation and function of osteoclasts can reveal the identification of new targets in the therapy of osteoclast-associated bone loss, a common feature of inflammation related diseases.
In this context, Tpl2 and Nalp3 protein are involved, in different ways, in inflammatory conditions. Tpl2 as a serine/threonine kinase of the MAP3Ks family, that is activated downstream of TLRs (TLR2,3,4,7,9) and TNFR (TNFR1 and CD40), that are important for the immune response and inflammation. ERK kinase, that is phosphorylated downstream of Tpl2, is implicated in three major steps of osteoclast differentiation: RANK/RANKL interaction, c-fms/M-CSF binding and integrins signaling pathway. To this end, it is important to delineate the role of Tpl2 in different cell types, such as osteoclasts, that are closely linked to the immune response. Nalp3 protein is involved in the immune response, through its participation in the formation of the multi-protein complex of inflammasome, that mainly mediates the production of IL-1b and IL-18. Osteoclasts are highly influenced, and positively affected in terms of differentiation and activation, by pro-inflammatory cytokines, and most importantly IL-1 and TNFa. Thus, the manipulation of Nalp3 could possibly affect the differentiation or function of osteoclasts.
Collectively, the data from the literature presented above led us to formulathe the hypothesis that Tpl2 and Nalp3 proteins could be implicated in the differentiation process towards osteoclasts. To this end, osteoclasts were differentiated from bone marrow cells isolated from Tpl2-/-, Nalp3-/- and wild type mice and the differentiation process was examined based on the osteoclast-specific TRAP staining, with the assesement of ERK phosphorylation on Tpl2-/- osteoclasts and mainly with the analysis of mRNA levels of osteoclast-specific genes in all three mouse strains. From the results obtained, it was apparent that not significant differences in osteoclast formation appeared in the two knock-out mouse strains. Phosphorylation of ERK was not different in Tpl2-/- mice as
compared to WT, upon the activation of osteoclasts with LPS or RANKL. Next, and most importantly, from the real-time PCR data, it was clearly shown that the levels of expression of osteoclast-specific genes, were comparable between all three mouse strains and for all genes studied (TRAP, NFATc1, MMP9 and cathepsin K). Subsequently, it was concluded that neither Tpl2 nor Nalp3 proteins are involved in the differentiation process of osteoclasts, since osteoclasts formation was not impaired in both Tpl2-/- and Nalp3-/- knock-out mice, and this was examined in different stages of the differentiation process.
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The role of Tpl2 and Nalp3 proteins in the process of osteoclastogenesis
