Taken together, these data demonstrate that MEK/ERK phosphorylation is the main pathway activated in human osteoblasts by INSL3. This finding fits well with the known effect of MAPK signaling in osteoblast proliferation/differentiation, and further supports our previous results. We also examined the Wnt/b-catenin signaling pathway, one of the most extensively studied pathways with direct relevance to basic bone biology. The importance of this pathway in bone formation is undeniable, and it has been demonstrated that the activation of the b-catenin signaling leads to increase bone mass, while suppression results in bone loss. b-catenin could be phosphorylated by GSK3b at S33/S37/T41 or by other kinases as PKA at S675 and Akt at S552. Furthermore, it has been recently reported a possible link between INSL3 and bcatenin during gubernaculum development. However, we excluded this pathway, as our data indicated that INSL3 does not influence b-catenin phosphorylation in human osteoblasts. The different results obtained by Kaftanovskaya in gubernacular cells could be due to a BAY 43-9006 supply crosstalk of pathways that are not involved n bone metabolism or to differences between the two different species. We next evaluated whether INSL3 stimulates genes involved in osteoblast proliferation/differentiation, matrix deposition and osteoclastogenesis. Other than an important effect on ALP production, we observed a significant effect of INSL3 on the expression of genes involved in the mineralization process, such as COL1A1, COL6A1, osteonectin, osteopontin, and TGF-b. The interaction among osteogenic-related molecules such as COL1A1 is well known as being related to matrix mineralization and COL6A1 has been suggested to play an important role in matrixmatrix interaction and in the construction of the extracellular structure. In some cases of osteoporosis, type VI collagen significantly decreases in bone suggesting that type VI collagen may be important for osteoid structure of bone. Osteonectin is the most abundant non-collagenous protein of developing bone and its high levels in forming bone may reflect a high proliferative potential of the functional osteoblast. Osteopontin comprises about the 2% of the non-collagenous protein in bone and it has important roles in bone turnover serving as attachment for osteoclasts activating the resorption cascade. TGF-b is produced by osteoblasts and regulates the proliferation and differentiation of osteoblasts both in vitro and in vivo by regulating the production of different genes such as those of the bone specific extracellular matrix proteins including type I collagen. INSL3 could therefore have an important role in matrix deposition as it stimulates the expression of genes coding for collagenous and non–collagenous proteins. These data well agree with the finding that INSL3-stimualted osteoblasts are fully differentiated and are able to mineralize the extracellular matrix. Furthermore, the increased expression of osteonectin and osteopontin underlines the effect of this hormone in the cellular proliferation process. We previously reported also a reduced osteoclasts population in Rxfp22/2 mice, so here we analyzed the effects of INSL3 on osteoblasts/osteoclasts crosstalk. With its membrane-bound osteoclast receptor RANK, while M-CSF stimulates osteoclastogenesis by binding with the c-FMS receptor on osteoclast surface.