Regulation Of SDF-1 Research Articles

GLI1 interaction with p300 modulates SDF1 expression in cancer-associated fibroblasts to promote pancreatic cancer cells migration.

Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300-GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.

Dipeptidyl Peptidase-4 Regulation of SDF-1/CXCR4 Axis: Implications for Cardiovascular Disease.

Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed protease that regulates diverse number of physiological functions. As a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (P1) amino acid residue from the amino terminus. The evidence to date supports an important effect of DPP4 in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which DPP4 inhibition improves glycemic control. DPP4 also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. In the current review, we will summarize the importance of DPP4-mediated enzymatic processing of cytokines/chemokines with an emphasis on SDF-1 and resultant implications for cardiovascular physiology and disease.

MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αvβ3-mediated SDF-1/CXCR4 signaling

The majority of glioblastoma multiforme (GBM) tumors recur after radiation (IR) treatment due to increased angiogenesis and IR-induced signaling events in endothelial cells (ECs) that are involved in tumor neovascularization; however, these signaling events have yet to be well characterized. In the present study, we observed that IR (8 Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, ECs treated with tumor-conditioned media (CM) obtained from IR-treated 4910 and 5310 cells showed increased microtubule formation. In view of this finding, we investigated the possible anti-angiogenic effects of MMP-2 downregulation using siRNA (pM.si) in IR-treated cells. We also determined the effect of CM obtained from mock, pSV (scrambled vector) and pMMP-2.si on endothelial cell growth and vessel formation. pM.si-CM-treated ECs showed inhibited IR-CM-induced SDF-1, CXCR4, phospho-PI3K and phospho-AKT and αvβ3 expression levels. In vitro angiogenesis assays also showed that the pM.si+IR decreased IR-induced vessel formation in ECs. Immunofluorescence and immunoprecipitation experiments indicated the abrogation of αvβ3-SDF-1 interaction in pM.si-CM-treated ECs when compared to mock or pSV treatments. External supplementation of either rhMMP-2 or rhSDF-1 counteracted and noticeably reversed pM.si-inhibited SDF-1, CXCR4, phospho-PI3K and phospho-AKT expression levels and angiogenesis, thereby confirming the role of MMP-2 in the regulation of αvβ3-mediated SDF-1/CXCR4 signaling. In addition to the in vitro results, the in vivo mouse dorsal air sac model also showed reduced angiogenesis after injection of pM.si alone or in combination with IR-treated xenograft cells. In contrast, injection of mock or pSV-treated cells resulted in robust formation of characteristic neovascularization. Collectively, our data demonstrate the role of MMP-2 in the regulation of SDF-1/CXCR4 signaling-mediated angiogenesis in ECs and show the anti-angiogenic efficacy of combining MMP-2 downregulation and IR when treating patients with GBM in the future.

Expression of Wnt-Inhibitors and SDF-1 in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma.

AbstractAbstract 2922Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by a combination of osteoclast hyperactivation and osteoblast inhibition. Several studies have shown that the Wnt-inhibitor DKK1 is one of the factors involved in osteoblast inhibition (Tian, NEJM, 2003), while the role of other Wnt-pathway inhibitors, such as secreted frizzled related protein-2 (SFRP2), SFRP3, sclerostin and Wnt inhibitory factor 1 (WIF1) are less studied. In addition stromal derived factor 1 (SDF-1/CXCL12) that interacts with CXCR4 in bone-marrow (BM) is a potential factor in OBD, although conflicting data has been reported. SDF-1has in mesenchymal stromal cells (MSC) been found downregulated in MM patients compared to healthy volunteers (HV) and also downregulated in MM plasma cells vs. normal plasma cells, while SDF-1 serum levels has been correlated to the extent of OBD in MM.Until now, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy whereby BM biopsies are snap-frozen and subsequently gene expression is analyzed. Using this strategy we have examined the gene-expression of Wnt inhibitors and SDF1. In addition, we have evaluated the protein levels of DKK1 and SDF-1 in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for DKK-1 (RnD, Quantikine) and SDF-1 (RnD, Quantikine) were run in duplicates according to manufacturer's instructions.Gene expression of DKK1 and SFRP3 were significantly (p<0.05) associated with OBD with increasing gene-expression with increasing OBD, while SDF-1 were significantly associated with decreasing gene-expression with increasing OBD. Gene expression of SFRP2, sclerostin and WIF1 did not associate with the extent of OBD. When comparing HV, MGUS and MM independent of OBD, sclerostin showed a tendency to be higher expressed in MM vs. MGUS and HV (p=0.08).A significant correlation between gene expression levels and protein expression levels was found for DKK-1 (Spearman's rho= 0.57, p=0.0006,) while the correlation of gene and protein expression levels of SDF-1 did not reach significance (Spearman's rho =0.31, p=0.074).The protein level of DKK1 in BM plasma was upregulated in MM and associated significantly with the OBD level (p<0.05). The protein level of SDF1 did not correlate to the extent of OBD.Our gene expression study of MM with minimal manipulation of the samples showed that the Wnt-inhibitors DKK1 and SFRP3 were significantly associated to the extent of OBD, while SDF1 gene expression inversely correlated with the extent of OBD. The report of simultaneous measurement of not only DKK1 but also SFRP3 and other Wnt-inhibitors is novel. The use of whole snap-frozen BM biopsies is a new method to evaluate gene expression in MM that makes it possible to include the tumor micro-environment and investigate patients independent of degree of MM plasma cell infiltration, and we were able to reproduce the DKK1 findings by others. The gene expression results were complemented by protein data for DKK1 and SDF-1. Our study shows for the first time the difference between SDF1 gene and protein levels in matched MM patient samples, and the difference measured incites us to further investigate the post-translational regulation of SDF-1, and the possibility of SDF1 protein being less bound to CXCR4 in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 197: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction

Myocardial infarction (MI) is the primary cause of death throughout the United States. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms may offer novel approaches for G-CSF-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter determined by echocardiogram in WT mice. No improvement on heart function is observed in Plg -/- mice, indicating Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to the ischemic area, BM transplantation with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP + cKit + cells (by 12 fold) in WT mice but not in Plg -/- mice. In addition, BM stem cell-derived vessels and arteries are infrequent in Plg -/- mice suggesting that Plg enhances cardiac repair by promoting stem cell recruitment to the lesion. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in the infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg -/- mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.

Abstract 13: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction

Ischemic heart disease, including myocardial infarction (MI), is the primary cause of death throughout the US. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms regulating G-CSF-induced cardiac repair may offer novel approaches for strengthening stem cell-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac tissue repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter by echocardiogram in wild-type mice. No improvement in tissue repair and heart function by G-CSF is observed in Plg -/- mice, indicating that Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to ischemia area, bone marrow transplantion (BMT) with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP+ cells (by 16 fold) in WT mice but not in Plg -/- mice, suggesting that Plg is essential for HPSC recruitment from BM to the lesion sites after MI. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg -/- mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.

Abstract 5277: MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αvβ3-mediated SDF-1/CXCR4 signaling

Abstract The majority of glioblastoma multiforme (GBM) tumors recur after the radiation (IR) treatment by increasing angiogenesis and IR-induced signaling events in endothelial cells that are involved in tumor neovascularization; these signaling events have yet to be well characterized. We observed that IR (8 Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, endothelial cells treated with tumor conditioned media (CM) obtained from IR-treated 4910 and 5310 cells showed increased microtubule formation. Thus, we investigated the possible anti-angiogenic effects of MMP-2 downregulation using siRNA (pMMP-2.si) in IR-treated cells and further determined the effect of CM obtained from mock, pSV (scrambled vector) and pMMP-2.si on endothelial cell growth and vessel formation. pMMP-2.si-CM-treated endothelial cells showed inhibited IR-CM-induced αvβ3, SDF-1, CXCR4, phospho-PI3K and phospho-AKT expression levels. In vitro angiogenesis assay showed that the pMMP-2.si+IR decreased the IR-induced vessel formation in endothelial cells. Immunofluorescence and immunoprecipitation experiments indicated the abrogation of αvβ3-SDF1 interaction in pMMP-2.si-CM-treated endothelial cells when compared to mock or pSV treatments. External supplementation of either rhMMP-2 or rhSDF-1 counteracted and noticeably reversed the pMMP-2.si-inhibited SDF-1, CXCR4, phospho-PI3K and phospho-AKT expression levels and angiogenesis, thereby confirming the role of MMP-2 in the regulation of αvβ3-mediated SDF-1/CXCR4 signaling. In corroboration with the in vitro results, the in vivo mouse dorsal air sac model also showed reduced angiogenesis with the injection of pMMP-2.si-treated xenograft cells whereas injection of mock or pSV-treated cells resulted in robust formation of characteristic neovascularization. Collectively, our data indicate the role of MMP-2 in the regulation of SDF-1/CXCR4 signaling-mediated angiogenesis in endothelial cells and demonstrates the anti-angiogenic efficacy of combining MMP-2 downregulation and IR when treating GBM in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5277. doi:1538-7445.AM2012-5277

SDF-1/CXCR4 signaling induces pancreatic cancer cell invasion and epithelial–mesenchymal transition in vitro through non-canonical activation of Hedgehog pathway

In our previous study, we found that blockade of SDF-1/CXCR4 signaling inhibits pancreatic cancer cell migration and invasion in vitro. However, the mechanism governing the downstream regulation of SDF-1/CXCR4-mediated invasion remains unclear. Here we report the role of SDF-1/CXCR4 in pancreatic cancer and the possible mechanism of SDF-1/CXCR4-mediated pancreatic cancer invasion. We show that there is a cross-talk between SDF-1/CXCR4 axis and non-canonical Hedgehog (Hh) pathway in pancreatic cancer. Furthermore, our data demonstrate that the ligand of CXCR4, SDF-1 induces CXCR4-positive pancreatic cancer invasion, epithelial–mesenchymal transition (EMT) process and activates the non-canonical Hh pathway. Moreover, we also demonstrate that the invasion of a pancreatic cancer and EMT resulting from the activation of SDF-1/CXCR4 axis is effectively inhibited by Smoothened (SMO) inhibitor cyclopamine and siRNA specific to Gli-1. Collectively, these data demonstrate that SDF-1/CXCR4 modulates the non-canonical Hh pathway by increasing the transcription of SMO in a ligand-independent manner. Taken together, SDF-1/CXCR4 axis may represent a promising therapeutic target to prevent pancreatic cancer progression.

Ion channels in the regulation of platelet migration

Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca2+ entry via Ca2+ channels. Ca2+ influx is sensitive to cell membrane potential which is maintained by K+ channel activity and/or Cl− channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca2+ activated K+ channel SK4 (sk4−/−) and their wild type littermates (sk4+/+). According to confocal microscopy human platelets expressed the Ca2+ channel Orai1 and the Ca2+-activated K+ channel KCa3.1 (SK4). SDF-1 (100ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10μM) and SKF-96365 (10μM), by unspecific K+ channel inhibitor TEA (30mM), by SK4 specific K+ channel blocker clotrimazole (10μM), but not by Cl− channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100μM). Significant stimulation of migration by SDF-1 was further observed in sk4+/+ platelets but was virtually absent in sk4−/− platelets. In conclusion, platelet migration requires activity of the Ca2+ channel Orai1 and of the Ca2+ activated K+ channel SK4, but not of NPPB-sensitive Cl− channels.

P53 Activation of Mesenchymal Stromal Cells (MSC) Partially Abrogates Microenvironment-Mediated Resistance to FLT3 Inhibition In FLT3/ITD AML

AbstractAbstract 2151Fms-like tyrosine kinase-3 (FLT3) inhibitors have recently been introduced to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3/ITD mutations. However, while ciculating blasts are rapidly eliminated, bone marrow (BM) responses are in general less impressive (Zhang et al., JCI 2009). One potential explanation for the reduced bone marrow response compared to the striking activity against circulating blast cells may be microenvironmental resistance to FLT3 inhibitors, including protection of FLT3/ITD+ blasts through the SDF-1 (CXCL12)/CXCR4 axis (Zeng et al., BLOOD 2009) and novel strategies to overcome microenvironmental resistance may further enhance the clinical benefit of FLT3 inhibitors. Here we investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3/ITD AML. We confirmed that Mdm2-p53 interaction and mutant FLT3 signaling in leukemic blasts were inhibited by the Mdm2 inhibitor Nutlin-3a and the selective FLT3 inhibitor FI-700, respectively, as previously reported (Kojima et al., BLOOD 2004; Kojima et al., LEUKEMIA 2009). BM samples were obtained from normal individuals or from FLT3/ITD AML patients with more than 70% leukemia cells after informed consent. BM-derived stromal cells were seeded in 12-well plates in MEM-alpha medium and exposed to Nutlin-3a for 24 hours, gamma-irradiated (2 Gy), or treated with 100 nM doxorubicin. The wells were then washed three times and AML cells were added. Cell cycle distribution was determined by propidium iodide staining and apoptosis by Annexin V binding. In FLT3/ITD AML cell lines and primary cells, apoptosis was induced by FI-700, but apoptosis induction was diminished under stromal coculture conditions (43.2 ± 0.5% versus 17.0 ± 1.2% in MOLM-13; p < .01). FI-700–induced reduction of Mcl-1 protein levels and activation of Bax were essentially abrogated when FLT3/ITD AML cells were co-cultured with MSC (20.9 ± 1.6% versus 8.3 ± 0.3% cells with activated Bax levels in MOLM-13;p < .01). Separation of MOLM-13 cells from stroma by 0.4 μM culture filters did not affect the protective effect of stromal cells (21.2 ± 2.3% versus 21.6 ± 1.6%). The protection appeared to be mediated partially by SDF-1/CXCR4 signaling because the addition of recombinant SDF-1 to control medium protected FLT3/ITD AML cells from FI-700–induced apoptosis while the addition of anti-CXCR4 antibody (R&D Systems) partially abrogated the protection conferred by stromal cells. The protective effect of stromal cells on MOLM-13 was significantly reduced when MSC were exposed to p53 inducers including Nutlin-3a (17.0 ± 1.2% versus 26.0 ± 1.8%; p < .01), gamma-irradiation (13.5 ± 3.0% versus 18.6 ± 3.1%; p < .05) or doxorubicin (14.2 ± 3.0% versus 19.7 ± 0.9%; p < .05), although p53 activation was not itself cytotoxic to stromal cells. However, SDF-1 mRNA and protein levels were reduced in p53-activated stroma cells. Our findings confirm that selective FLT3 inhibition may not eradicate FLT3/ITD AML cells protected by stroma and show, for the first time, that p53 activation in stroma cells blunts stroma cell-mediated resistance to a kinase, here a FLT3 inhibitor, in part through regulation of SDF-1. Results suggest that combinations of HDM2/p53 (R7112/Nutlin-3a, Andreeff et al. ASH 2010) and/or CXCR4 inhibitors (Plerixafor, Konopleva et al. ASH 2010) with FLT3 inhibitors should be investigated in clinical trials targeting FLT3 mutant leukemias, with the dual goal of inducing apoptosis in leukemic cells and, concomittantly, reduce the protective effects exerted by the marrow microenvironment. Disclosures:No relevant conflicts of interest to declare.

Estrogen-induced stromal cell-derived factor-1 (SDF-1/ Cxcl12) expression is repressed by progesterone and by Selective Estrogen Receptor Modulators via estrogen receptor α in rat uterine cells and tissues

Endometriosis, defined as the presence of endometrial glands and stroma at extra-uterine sites, is a gynecological condition that affects women of reproductive age. Consistent with its uterine origins, endometriotic lesions and resulting symptoms are hormonally responsive. To investigate Progesterone Receptor (PR)-based therapies, we measured physiological endpoints and gene expression in rat models of uterine cell estrogenic activity. Estrogen-induced ELT-3 rat leiomyoma cell proliferation was significantly inhibited by progesterone (P4), while the antiprogestin RU486 or the Selective PR Modulator (SPRM) asoprisnil, did not block proliferation. Stromal cell-derived factor-1 (SDF-1/ Cxcl12) gene expression was induced by estrogen, and was repressed by the Selective Estrogen Receptor Modulators (SERMs), the antiestrogen ICI 182,780, and P4, but not by RU486 or the ERβ-selective ligand ERB-041. In ELT-3 cells, asoprisnil demonstrated partial PR agonism on SDF-1 gene repression. Magnetic Resonance Imaging was used to monitor development of ectopic cysts in a rat surgical model of endometriosis. SERMs and P4 significantly decreased cyst volumes comparably by ∼60%. However, ERB-041 and asoprisnil had no effect on cyst volume, and RU486 increased cyst volume by 20%. SDF-1 expression was modestly, but significantly, increased in the cyst compared to eutopic uterus, and P4 and raloxifene could repress the expression. We showed that SDF-1 was similarly regulated in human cells. These data suggest that transcriptional regulation of SDF-1 is a surrogate marker of estrogenic activities via ERα in rat uterine cells, and that SDF-1 repression by PR agonists can predict the ability to oppose the actions of estrogen in vivo.

Functional SDF-1 Secretion from BM Stromal Cells Is a Cell Contact-Dependent Event Mediated by Cx43 and Cx45 Gap-Junctions

Introduction: Bone marrow (BM) supporting stromal cells secrete an array of important signaling factors, in a synchronized fashion, creating a unique microenvironment required for regulation of hematopoietic stem and progenitor cells (HSPC) maintenance, localization, self-renewal and development. The chemokine SDF-1 (also termed CXCL12), whose fine tuned levels are critical for HSPC quiescence, is the only known powerful chemotactic factor for both human and murine HSPC. BM stromal cells rapidly respond to alarm situations by secreting SDF-1 to the circulation, promoting progenitor cell mobilization (Dar et al., ASH 2006) as part of host defense and repair mechanism. This well synchronized event suggests that the supporting stromal cells function as a physiological syncytium. However, the mechanism governing the synchronized SDF-1 de-novo synthesis and secretion from BM stromal cells is not known. The gap-junction (GJ) intercellular channels protein Connexins (Cx), allow the passage of secondary messengers between cells in a tissue, thus synchronizing its activity. The existence of the multi-function Cx43 and Cx45 GJ proteins in the BM compartments suggests a direct intercellular communication, coordinating their supporting capacity.Results: In order to evaluate Cx networks involvement in the regulation of SDF-1 secretion, we first checked for Cx43 expression in the BM of mice treated with G-CSF. We have previously shown that as part of G-CSF induced mobilization, SDF-1 levels are downregulated in the BM, thus promoting HSPC proliferation and mobilization (Petit et al., Nat.Immunol, 2002). Cx43 was strongly downregulated both at mRNA and protein levels in the BM of G-CSF treated mice, in correlation with reduced levels of SDF-1. Hence, we asked whether Cx directly regulates SDF-1 secretion. Treating BM stromal cells with carbenoxolone (CBX), a known Cx blocker, resulted in immediate, complete and specific blockage of SDF-1 secretion to the conditioned medium (CM). Secretion of the cytokine HGF, measured as control, was not affected. In addition, prolonged stimulation of BM stromal cells with CBX resulted in elimination of Cx43 and Cx45 transcription, as well as SDF-1 transcription. We then evaluated the direct cell-contact necessity for SDF-1 secretion. Equal numbers of BM stromal cells were seeded in different-sized culture plates allowing and restricting cell contact, in the same volume of medium. SDF-1 levels in the CM were found to be significantly higher in the contacting BM stromal cell cultures compared to the non-contacting BM stromal cell cultures, while HGF levels were not affected. Surprisingly, similar SDF-1 mRNA levels were detected in both groups. These results suggest that SDF- 1 secretion, but not transcription, is a cell contact dependent event. Next, the functional consequences of stromal Cx blockage was assessed by testing migration of human G2 cells towards CM collected from non-treated and CBX-treated BM stromal cells. G2 cells failed to migrate towards CBX-treated BM stromal cells CM. This was further supported by in-vivo homing experiments - BM mononuclear cells were intravenously injected to PBS and CBX treated C57BL/6 recipients. CBX-treated mice exhibited reduction in homing capacities to the femoral BM, in correlation with lower BM levels of SDF-1. cAMP is a small secondary messenger transmitted by Cx between cells and thus may serve as the mediator of Cx regulated SDF-1 secretion. cAMP inhibition in BM stromal cells by MDL resulted in CBX-like effect - decreased SDF-1, Cx43 and Cx45 mRNA as well as decreased SDF-1 secretion. Interestingly, cAMP elevation in BM stromal cells, by dbcAMP or forskolin, resulted in reversed regulation of SDF-1 and Cx43/Cx45. cAMP effect on SDF-1 is PKA-independent since pretreatment of BM stromal cells with H89 did not impair the cAMP elevation effects.Conclusions: Here we show, for the first time, that functional SDF-1 secretion from BM stromal cells is a cell contact-dependent event mediated by Cx43 and Cx45 gap junctions. We have identified dual regulatory roles of BM-Cx on SDF-1: a gap junction-free regulation of its transcription and a gap junctiondependent regulation of its secretion. We hypothesize that Cx43/Cx45 channels transmit cAMP, which in turn controls synchronized SDF-1 secretion and subsequent stem cell migration and development events.

The Retinoblastoma Binding Protein RBP2 Is an H3K4 Demethylase

Changes in histone methylation status regulate chromatin structure and DNA-dependent processes such as transcription. Recent studies indicate that, analogous to other histone modifications, histone methylation is reversible. Retinoblastoma binding protein 2 (RBP2), a nuclear protein implicated in the regulation of transcription and differentiation by the retinoblastoma tumor suppressor protein, contains a JmjC domain recently defined as a histone demethylase signature motif. Here we report that RBP2 is a demethylase that specifically catalyzes demethylation on H3K4, whose methylation is normally associated with transcriptionally active genes. RBP2-/- mouse cells displayed enhanced transcription of certain cytokine genes, which, in the case of SDF1, was associated with increased H3K4 trimethylation. Furthermore, RBP2 specifically demethylated H3K4 in biochemical and cell-based assays. These studies provide mechanistic insights into transcriptional regulation by RBP2 and provide the first example of a mammalian enzyme capable of erasing trimethylated H3K4.

Regulation of SDF-1 (CXCL12) production by osteoblasts; a possible mechanism for stem cell homing

Stromal derived factor-1 (SDF-1 or CXCL12) controls many aspects of stem cell function including trafficking and proliferation. Previously, it was demonstrated that DNA-damaging agents such as irradiation, cyclophosphamide or 5-fluorouracil increase the expression of SDF-1 by osteoblasts in murine marrow. Here, the production of SDF-1 by osteoblasts in vitro in response to cytokines known to be particularly important in bone physiology was examined using primary human osteoblasts (HOBs), mixed marrow stromal cells (BMSCs), and by, mouse, rat and human osteoblast-like cell lines. From these studies, it was determined that the expression of SDF-1 is an early feature of osteoblastic induction that may be modulated by IL-1β, PDGF-BB, VEGF, TNF-α and PTH. Each of these factors increased SDF-1 synthesis, while TGF-β 1 decreased SDF-1 secretion. Of note, the biodistribution of SDF-1 in culture was equally distributed between the medium and detergent-soluble and -insoluble fractions of the cultures. Immunohistochemistry of developing bones demonstrated that SDF-1 was also a feature of early bone development first beginning in the perichondrium and moving into the marrow cavity of the developing bone analogue. As SDF-1 expression increases in response to PTH in vitro, animals were treated with an anabolic regime of PTH for 21 days. Under these conditions, significant increases in SDF-1 mRNA expression were observed near the growth plate and epiphysis regions of the long bones. Yet, in serum, immunodetectable SDF-1 levels were significantly reduced (24%) in the PTH-treated animals (Vehicle: 408 ± 25 vs. PTH 308 ± 20 SDF-1 pg/ml). Together, these data suggest a possible mechanism for localizing stem cells into a developing marrow where increased expression of SDF-1 in the local marrow environment along with decreased SDF-1 in the serum may create a homing gradient.

Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1.