Success of transplantation significantly depends on the quality and functionality of donor HSCs. Consequently , use of pharmacological agents to improve engraftment of donor HSCs is being aggressively pursued. In the present study, we examined the result of NO donors on the engraftment ability of murine HSCs. We also elucidated the molecular mechanisms active in the process. Here, we show for the first time that NO induce CD34 expression in LSK-CD34
HSCs and this has a contrasting age-dependent effect on their functionality (Fig.
Graphical summary. SIMPLY NO nitric oxide
In a murine system, CD34 antigen manifestation is considered as a marker of short-term engrafting HSCs (ST-HSCs). However , previous reports have demonstrated that the expression associated with CD34 on murine HSCs is context-dependent, and has 2 implications: it could either be an indication of progressive dedication and differentiation giving rise to ST-HSCs or it might indicate an activation status of LT-HSCs, as demonstrated in 5-fluorouracil-treated mice [ 43 ]. It has also been demonstrated that CD34 + HSCs gathered from young mice (6– 8 weeks old; juvenile HSCs) possess engraftment capacity, but the CD34 − do not, while in adult mice (> 10 several weeks old; adult HSCs) the situation is reversed [ 24 ]. These data show that CD34 expression has age- as well as context-dependent implications in murine hematopoiesis. In our research, we found that SNP-treated juvenile HSCs engraft more proficiently, whereas their adult counterparts do not. Thus, our information provide direct evidence for the context-dependent role of CD34 in functionality of murine HSCs.
In vitro exposure of murine BM tissue (12 weeks old) to NO donors has been documented to induce myeloid commitment of HSCs. Similarly, remedying of adult mice with NO donors resulted in expansion of HSCs in their bone marrow, but these HSCs lost their extensive engraftment capacity [ 26 ]. Although these results were effective of an increase in ST-HSCs, whether LSK-CD34 − LT-HSCs acquired CD34 expression in response to NO plus contributed to the ST-HSC population, or whether LSK-CD34 + cells themselves proliferated in response to SIMPLY NO, was not investigated. Also, previous studies did not distinguish between the consequence of NO on juvenile as compared to adult HSCs. Such a variation is important since CD34 expression has been shown to have development-specific ramifications in the functionality of murine HSCs [ 24 ].
In the present study, we identified murine CD34 and LSK-CD34 − cellular material as the molecular and cellular targets, respectively, of SIMPLY NO signaling. We demonstrated that NO induces CD34 surface area expression and Cd34 mRNA expression in LSK-CD34 − cells in a time- and dose-dependent manner. It was also supported by a strong correlation between the upregulation associated with CD34 and the nitrite concentrations in the medium (an roundabout measure of NO). These data clearly establish that the NO-mediated increase in LSK-CD34 + cells relates to acquisition of CD34 expression by the LSK-CD34 − HSCs, rather than proliferation of LSK-CD34 + HSCs.
Not many reports describe the mechanisms involved in the regulation of CD34 expression in murine HSCs. The transcription start websites of both murine and human CD34 genes consist of consensus binding sites for several transcription factors, three which, myb, ets and mzf-1, have been shown to affect CD34 expression [ 44 – 46 ]. Sp1 has been shown to regulate murine CD34 at the transcriptional level [ 33 ]. To the best of our knowledge, our bait is the first report showing that NO regulates CD34 expression in murine HSCs at both the transcriptional as well as the translational levels.
NO is recognized to regulate several TFs [ 47 ], including c-Myb and c-Jun . Both these TFs play a significant role in HSC proliferation and are also known to regulate CD34 expression in various cell systems [ 37 , 48 , 49 ]. Using siRNA-mediated gene silencing, we demonstrated that the NO-mediated upregulation associated with CD34 is dependent on c-Myb as well as c-Jun. Silencing of c-Jun had a stronger impact on NO-mediated CD34 expression in the LSK CD34 − HSCs, as compared to silencing of c-Myb , suggesting that c-Jun plays a crucial function in the process. The milder effects seen with silencing associated with c-Myb are usually consistent with the work published by Krause et al. [ 50 ]. Upregulation of mitochondrial biogenesis has been shown to seite an seite upregulation of CD34 in murine HSCs [ 51 ]. Whether NO upregulates mitochondrial biogenesis in the murine HSCs remains to be seen.
The most important finding in this particular study was that the NO-mediated upregulation of CD34 within juvenile HSCs (6– 8 weeks) boosted their engraftment capacity. In sharp contrast, such an upregulation of CD34 in the adult HSCs (10– 12 weeks) significantly decreased their engraftment capacity. These differences in the engraftment capabilities of juvenile and adult CD34 + HSCs are similar to previous reports showing that CD34 + HSCs from juvenile rodents, but not from adult mice, possess engraftment capacity [ 24 , 25 ]. High levels of c-Myc are known to compromise the particular self-renewal ability of HSCs [ 28 ]. We discovered that the NO-mediated upregulation of CD34 was accompanied by a solid upregulation of c-Myc in adult HSCs, but not in juvenile HSCs. Similarly, the expression of Pu. 1 , a myeloid commitment marker, has been highly upregulated only in the adult HSCs. These information indicate that NO-induced CD34 expression is accompanied by a good induction of the differentiation program in adult HSCs.
The contrasting functionality observed in teen compared to adult HSCs could also be correlated with the differential legislation of some important TFs by NO in the 2 types of HSCs. The expression of specific TFs chosen for their binding to the CD34 promoter, their involvement within NO signaling in various systems, and their importance within the hematopoietic system was examined in NO-treated HSCs through juvenile and adult mice. Yy1 , a multifunctional polycomb group (PcG) transcribing factor, has been shown to mark phenotypically defined and practical LT-HSCs and is also known to interfere with the commitment process [ 30 ]. Consistent with these reports, Yy1 was found to be significantly upregulated by NO in juvenile HSCs, but not in grownup HSCs. The regulation of Cd34 transcription requires Sp1 [ 33 ]. We discovered that NO upregulated Sp1 in both types of HSCs, but in juvenile HSCs the levels of Sp1 were significantly higher. However , the level of Cd34 in NO-treated teen HSCs was significantly lower as compared to that in NO-treated adult HSCs. TF-bind software-based analysis showed that Yy1 binds towards the Cd34 marketer, but its role in the regulation of Cd34 has not been elucidated. YY1 is really an ubiquitously distributed transcription factor that is involved in repressing plus activating a diverse number of promoters. It would be interesting to find out whether it negatively regulates CD34 expression, thereby countering the positive regulatory effects of Sp1 on Cd34 in juvenile cells. Yet another TF which was strongly upregulated by NO in juvenile HSCs had been Chop1 . It is a simple leucine-zipper protein induced by a variety of stimuli, including SIMPLY NO [ 52 ], that evokes cellular stress responses. However, deletion of its homologous TF, C/EBP, has been shown to have an effect on self-renewal of HSCs [ 31 ]. It is thus probable that, in the juvenile HSCs, Chop1 acts as a self-renewal factor and increases the engraftment ability of the HSCs. Consistent with previous reports displaying Runx-1 as being a marker for long-term engrafting HSCs [ 32 ], we all found that Runx1 levels in NO-treated juvenile HSCs were considerably higher as compared to their adult counterparts. Thus, high amounts of Yy1 , Chop1 , and Runx1 , coupled with a comparatively lower amount of Cd34 , may have led to the self-renewal and increased functionality of juvenile HSCs. Our findings emphasize the age-specific influences of SIMPLY NO in filling an important gap in the existing knowledge about the role in hematopoiesis.
The top expression of CXCR4 on HSCs plays an important function in their homing, engraftment, and retention in the bone marrow of recipients [ 53 ]. NO has been shown to enhance CXCR4 expression in human CD34 + HSCs collected from adult (BM and mobilized PB) as well as neonatal sources (CB). Taking into consideration the differences in engraftment shown by NO-treated juvenile and adult HSCs, possible exists that this may have been due to age-dependent effects of NO upon CXCR4 expression in murine HSCs. However , we discovered that the treatment of juvenile as well as adult murine HSCs without resulted in a strong upregulation of CXCR4 on their surface, judgment out the possibility that the differential effect of NO on their hair transplant ability could be related to differential effects on CXCR4 surface area expression. Treatment of murine HSCs with SNP has been shown in order to abrogate PGE2-mediated upregulation of CXCR4 [ 10 ]. Nevertheless , we found that SNP increases CXCR4 in both mature and juvenile HSCs. It is possible that a combined presence associated with SNP and PGE2 negatively regulates CXCR4. Our information showing compromised engraftment of SNP-treated adult HSCs regardless of elevated CXCR4 expression suggest that a mere upregulation of CXCR4 surface expression cannot be taken as a reliable indicator of engraftment ability of the HSCs.
Our own data could have relevance in clinical transplantation. Use of medicinal compounds to enhance homing and engraftment of HSCs continues to be widely attempted for clinical transplantation. Such manipulation gets important when the HSCs are either fewer in amounts (fetal liver, CB) or possess compromised quality (aged donors). Our murine data, taken together with the preliminary information generated with CB and MBPL cells, show that will NO exerts age-dependent effects on HSC functionality.
In a wider perspective, our information point towards a possibility that several other pharmacological reagents could also have such contrasting effects on the functionality of HSCs from developmentally distinct sources, and therefore HSC age might be considered as an important parameter while screening various compounds for potential use in HSC manipulation.