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    • It has been demonstrated that CCR

    2018-10-29

    It has been demonstrated that CCR1 is biologically functional in BMSCs (Honczarenko et al., 2006). Here we have shown that CCL5 induces biological responses via CCR1, indicating that the CCL5/CCR1 axis is functional also in ADSCs. CCL5 may also bind to the CCR3 and CCR5 receptors; however, according to our data, expression of these receptors is very low or not detectable in ADSCs. We show that CCL5 signaling leads to activation of the NFκB pathway, which is known to be involved in the regulation of cell proliferation, survival, migration and invasion processes (Hayden and Ghosh, 2012; Wu and Zhou, 2010). NFκB transcriptional activity as well as expression of several NFκB target genes is altered in ADSCs in response to CCL5 treatment. We demonstrate rapid up-regulation of genes encoding endopeptidases (MMP1, MMP2 and MMP9) that hydrolyze the components of ECM (e.g. collagen, fibronectin, laminin), thus contributing to migratory and wound healing processes (John and Tuszynski, 2001). Up-regulation of MMP points to the ability of ADSCs to re-modulate ECM in response to CCL5 stimulation. Furthermore, it is an indication of activation of migratory mechanisms in ADSCs demonstrated by cell migration assay here and by others (Baek et al., 2011). Nevertheless, further analyses are needed to elucidate, whether CCR1+ ADSCs are also capable of chemotaxis in vivo and whether these cells are able to differentiate and contribute to processes of tissue regeneration. The NFκB pathway has been proposed to trigger pro- and anti-apoptotic signals in different cells (Chu et al., 1997; Ryan et al., 2007). Here we show that the NFκB pathway activated upon CCL5 stimulation rather inhibits cell death in ADSCs. CCL5 signaling suppresses the expression of pro-apoptotic genes BID and BAXα, whereas expression of SURVIVIN, an inhibitor of apoptosis and important positive regulator of histamine-1 receptor antagonist activated in G2/M phases, increases (Chandele et al., 2004). Moreover, we show that a number of BrdU+ and CCR1+ cells increases in response to CCL5 signaling after 18 and 48h of treatment initiation, respectively. Taken together, these data indicate that CCL5 induces proliferation of CCR1+ ADSCs. It has been suggested that molecular mechanisms regulating the undifferentiated state of stromal stem/progenitor cells from different tissues are related to the expression of distinct transcription factors, such as OCT4, SOX2 and NANOG (Greco et al., 2007; Park et al., 2011; Saulnier et al., 2011). Down-regulation of these factors is associated with loss of multipotency and self-renewal, and is proposed to state the beginning of subsequent differentiation steps (Meshorer and Misteli, 2006; Ng and Surani, 2011; Pan et al., 2006). Here we show that OCT4, SOX2 and NANOG are expressed in ADSCs; however, expression of SOX2 has not been detected in some pools of ADSCs, distinguished by the lower expression of other multipotency genes and CCR1. Interestingly, the expression levels of OCT4, NANOG and SOX2 demonstrated statistically significant correlation with the expression levels of CCR1 in ADSC samples. Linear regression analysis has revealed that the coefficients of determination (R2) obtained for the levels of CCR1 and multipotency gene expression are comparable with that of multipotency genes, with SOX2/NANOG demonstrating the highest R2=0.98. Moreover, the levels of multipotency gene expression were elevated in concert with up-regulation of CCR1 expression upon stimulation of ADSCs with CCL5. There are at least two possible explanations of this phenomenon. First, the transcriptional up-regulation of multipotency genes via factors unidentified in the present study may occur at a single cell level. Second, since the increase in the gene expression levels is detected following 48h of treatment initiation, it is more likely that higher mRNA and protein levels occur due to the increased amount of CCR1+ cells demonstrated here. These data suggest that one of the features of “real” adipose tissue derived stem/progenitor cells expressing OCT4, NANOG and SOX2 may be the expression of CCR1 receptor. The presented data suggest that the CCL5/CCR1 axis participates in the regulation of ADSC stemness and multipotency, and consequently may have an impact on differentiation of ADSCs. Also, data presented here suggest a possible method for separating populations of ADSCs possessing higher stemness-related properties by exploiting the expression of CCR1 receptor.