ABSTRACT
Objective: receptor activator of nuclear factor-kappa B ligand [RANKL] appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells [HSCs]. This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor [CTR] on cord blood HSC surface
Materials and Methods: in this experimental study, CD133[+] hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor [M-CSF] and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase [TRAP] staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction [RT-PCR] assay for specific genes
Results: hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells
Conclusion: presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast
ABSTRACT
Multiple myeloma [MM] is a plasma cell malignancy where plasma cells are increased in the bone marrow [BM] and usually do not enter peripheral blood, but produce harmful factors creating problems in these patients [e.g. malignant plasma cells over activate osteoclasts and inhibit osteoblasts with factors like RANKL and DKK]. These factors are a main cause of bone lesion in MM patients. Recently SOST gene which responsible to encodes the sclerostin protein was identify. This protein specifically inhibits Wnt signaling in osteoblasts [inhibition of osteoblast differentiation and proliferation] and decrease bone formation and can also cause bone lesion in MM patients. In this experimental study, human myeloma cell lines [U266 b1] were purchased from Pasteur Institute of Iran. Samples consisted of BM aspirates from the iliac crest of MM patients. BM with more than 70% plasma cell were selected for our study [6 patients] and one healthy donor. RNA extraction was done with Qiagen kit. was undertaken on mRNA of samples and cell lines. Also we purchased unrestricted somatic stem cells from Bonyakhte Company to evaluate the effect of soluble factors from myeloma cell lines on osteogenic differentiation medium. Our results showed that SOST is expressed significantly in primary myeloma cells derived from MM patients and myeloma cell lines. In other words, patients with more bone problems, express SOST in their plasma cells at a higher level. In addition, myeloma cells inhibit osteoblast differentiation in progenitor cells from umbilical cord blood stem cell [UCSC] in osteogenic inducing medium. There are many osteoblast maturation inhibitory factors such as DKK, Sfrp and Sclerostin that inhibit maturation of osteoblast in bone. Among osteoblast inhibitory agents [DKK, Sfrp, Sclerostin] sclerostin has the highest specificity and therefore will have less side effect versus non-specific inhibitory agents. Our results also show that based on SOST expression in MM, there is a potential to inhibit sclerostin with antibody or alternative methods and prevent bone lesion in MM patients with the least side effect