HS27a Cells Research Articles

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.

A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine whose levels in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. Here, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function and therefore contributes to OB/OC uncoupling in MM.

Establishment of An HS23 Stromal Cell-Dependent Myeloma Cell Line: Fibronectin and IL-6 Are Critical for Survival

AbstractAbstract 2949Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells triggers cytokine-mediated tumor cell growth, survival, and drug resistance. In particular, integrin a4b1, very late antigen 4 (VLA-4)-mediated fibronectin adhesion confers a survival advantage to myeloma cells. One of the problems in treating patients with MM is that it is very hard to eliminate residual myeloma cells, even following high-dose chemotherapy followed by auto-stem cell transplantation. Importantly, cell adhesion-mediated drug resistance (CAM-DR) must be overcome in order to eliminate the minimal residual disease of MM. Here we characterized a multiple myeloma cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first two months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and subsequently, they continued to proliferate on HS23 stromal cells but not on HS27A stromal cells (both stromal cells were established by Torok-Storb B, Blood 1995). The phenotype of the established MSG1 cell line was: CD138+, CD38++, CD19−, CD56−, VLA-4+, VEGFR1+, and VEGFR2+. Furthermore, immunohistochemical staining demonstrated expression of IgA and λ chain in the cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities, basically hypertriploidy including the deletion of chromosome 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured on fibronectin-coated plates with the supernatant of HS23 cells or RPMI1640 medium supplemented with 10% FBS (control medium) containing IL-6 (10 ng/ml). Notably, MSG1 could not survive in control medium containing IL-6 or in HS23 supernatant unless bound to fibronectin, which was also expressed on HS23 and HS27A cells. IL-6 and VEGF production were detected in the supernatants of both HS23 and HS27A stromal cells (36.8±4.5 pg/ml and 131±5.8 pg/ml; 13.2±1.9 pg/ml and 16664±418 pg/ml, respectively). Next, we analyzed the effect of tocilizumab, an anti-IL-6R antibody, and bevacizumab, an anti-VEGF antibody on MSG1 survival. Tocilizumab (50 μ g/ml) inhibited MSG1 survival when cultured on fibronectin-coated plates in control medium containing IL-6 (10 ng/ml), and tocilizumab (10 μ g/ml) inhibited MSG1 survival when cultured on HS23 stromal cells. However, bevacizumab (500 μ g/ml) did not show such inhibition. Therefore, MSG1 survival depends on HS23 stromal cells: in other words, it depends on binding to fibronectin and IL-6. If these factors induced CAM-DR in myeloma cells, MSG1 may be a unique myeloma cell line that will be useful for analysis of CAM-DR, and tocilizumab might be a useful drug for treatment of MM. Furthermore, since MSG1 could survive on irradiated HS27A cells, and since HS23 and HS27A express similar adhesion molecules (Torok-Storb B et al., Blood 1995), these data suggest that HS27A might secrete factors that are detrimental to MSG1 survival. The identification of such an inhibitory factors could be of interest in terms of the regulation of myeloma proliferation. Disclosures:No relevant conflicts of interest to declare.

High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-193a as a Regulator of Jagged1 In Marrow Stromal Cells.

AbstractAbstract 3847MicroRNAs (miRNAs) are small non-coding RNAs with important roles in the post-transcriptional regulation of up to 30% of all vertebrate genes. Traditional methods to determine miRNA-mRNA interactions have included transcriptional profiling of miRNAs, bio-informatic prediction of miRNA-mRNA binding, analysis of 3' untranslated region (3'UTR) binding of miRNAs and over-expression of miRNAs in relevant cell types. These studies however fall short of demonstrating direct interaction between a miRNA and its target mRNAs. We applied a recently described biochemical technique of high throughput sequencing following cross-linked immune precipitation (HITS-CLIP) to dissecting the miRNA-mRNA interactions in two functionally distinct human marrow stromal cell lines. HITS-CLIP relies on the ability of ultraviolet (UV) radiation to cross-link RNA to proteins they are bound to, followed by immune-precipitation of the RNA-protein complex to isolate the cross-linked RNA and sequencing by high throughput techniques. As miRNA-mRNA interactions occur in close proximity to the argonaute proteins (AGO), an anti-argonaute monoclonal antibody was used to isolate the Ago-miRNA-mRNA complexes.The two stromal cell lines analyzed by HITS-CLIP (designated HS5 and HS27a) were isolated from a normal marrow primary long term culture (LTC), immortalized and extensively characterized for both function and expression profiles (mRNA and miRNA). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (CXCL12, Angiopoietin-1, Jag1 etc). In keeping with this, HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. Sequence reads from the HITS-CLIP analysis from each of the cell lines were aligned to the human genome using the UCSC genome browser to identify Ago-mRNA and Ago-miRNA binding sites in both the cell lines. Interestingly, corresponding datasets from HS5 and HS27a were similar for the majority of mRNAs and miRNAs, but distinct for those mRNAs (such as Jag1, CXCL12, IL6 and GCSF) and miRNAs (such as miR-886-3p, miR-221, miR-181a and miR-193a) known to be differentially expressed between the two cell lines. We then validated the use of the HITS-CLIP strategy in stromal cells by analyzing one such Ago-mRNA binding site for Jagged1 (Jag1). Jag1 is a ligand for Notch1 and is expressed in those cells that support the hematopoietic stem cell (HSC) niche. The Notch pathway is a highly conserved signaling system critical in regulating several tissue systems including hematopoietic cells. This binding site, 1749 bp downstream of the transcriptional start-site for Jag1 was significantly more enriched in HS5 compared to HS27a. The site was also a predicted binding site for miR-193a, a miRNA over-expressed in HS5 compared to HS27a cells. Over-expression of miR-193a in HS27a cells resulted in the down-regulation of Jag1 protein (as measured by Western blotting). To confirm the direct interaction between Jag1 and miR-193a, we cloned this purported binding site downstream of the luciferase gene and co-transfected the plasmid with miR-193a. Luciferase activity was down-regulated greater than 50% when compared to control transfections suggesting a direct effect of miR-193a on Jag1 transcript. In summary, our data suggest that HITS-CLIP methodology can be used to define in vivo spatial interactions between miRNA and mRNAs in the marrow microenvironment (ME). It can also be used to define miRNA-based regulation of specific genes such as Jag1, which are critical to defining functional niches in the ME. Disclosures:No relevant conflicts of interest to declare.

Mir-886-3p Contributes to the Regulation of the hematopoietic microenvironment by down-Regulating SDF-1α (CXCL12).

Abstract 561Two functionally distinct stromal cell lines were isolated from a primary long term culture (LTC) established from aspirated human marrow. Designated HS5 and HS27a, the lines were immortalized and extensively characterized including expression profiles for both messenger (mRNA) and micro-RNA (miRNA, a recently described class of small non-coding RNAs that regulate gene expression by binding to target mRNAs to prevent their translation). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (SDF-1αa, angiopoietin-1 etc). In keeping with this HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. When cultured together to better mimic in vivo cell-cell interactions, the gene expression of HS27a and HS5 combined differed from the expected sum of the two parts, exemplified by the 5-fold down regulation of SDF-1α.Comparisons of miRNA expression profiles of HS5 and HS27a determined that mir-886-3p, (previously described by deep sequencing of small RNA libraries) was expressed > 40 fold in HS5 compared to HS27a, this was then confirmed by quantitative RT-PCR. Given the abundance of mir-886-3p and the possibility that it could be secreted by HS5 and taken up by cells in contact with HS5, we tested its effect on gene expression in HS27a. Transcript levels of genes associated with the stem cell niche (Jagged1, BMP4, Angiopoietin-1, SDF-1α, VCAM-1 and N-Cadherin) were determined by quantitative RT-PCR after direct transfection of mir-886-3p precursors into HS27a cells and compared to appropriate controls. Results show SDF-1α mRNA expression was down-regulated by as much as 8 fold 3 days after transfection. Levels of secreted SDF-1α in culture media, as determined by ELISA, were also decreased. Since SDF-1α is a chemokine known to be critical for the homing of hematopoietic stem and progenitor cells to their niche, the functional significance of the SDF-1α down-regulation by mir-886-3p was confirmed by decreased chemotaxis of T-lymphocytic cells (Jurkat) following miRNA transfection of stromal cells. To determine if mir-886-3p directly effects the SDF-1α transcript, the 1.5 kbp 3'untranslated region (UTR) of SDF-1α gene was cloned downstream of the luciferase gene, and co-transfected with mir-886-3p into HS27a cells. Results showed the luciferase activity was down-regulated greater than 50% in the presence of mir-886-3p, suggesting a direct effect on the SDF-1 α transcript.Given the concern over the relevance of immortalized cell lines we investigated Mir-886-3p expression in primary marrow stromal cells at early passage sorted on the basis of +/- expression of CD146. (CD146 or MCAM has been reported to define a population that supports the hematopoietic stem/ precursor cell niche and is expressed by HS27a and not HS5 cells). Results indicated that the CD146+ stromal cells had significantly lower expression of mir-886-3p when compared to CD146- cells.In summary, these data suggest a role for miRNA in modulating the expression of gene products that are associated with the hematopoietic stem cell niche. Disclosures:No relevant conflicts of interest to declare.

Tumour necrosis factor‐induced gene expression in human marrow stroma: clues to the pathophysiology of MDS?

Aberrant regulation of the tumour necrosis factor alpha gene (TNF) and stroma-derived signals are involved in the pathophysiology of myelodysplasia. Therefore, KG1a, a myeloid leukaemia cell line, was exposed to Tnf in the absence or presence of either HS-5 or HS-27a cells, two human stroma cell lines. While KG1a cells were resistant to Tnf-induced apoptosis in the absence of stroma cells, Tnf-promoted apoptosis of KG1a cells in co-culture experiments with stroma cells. To investigate the Tnf-induced signals from the stroma cells, we examined expression changes in HS-5 and HS-27a cells after Tnf exposure. DNA microarray studies found both discordant and concordant Tnf-induced expression responses in the two stroma cell lines. Tnf promoted an increased mRNA expression of pro-inflammatory cytokines [e.g. interleukin (IL)6, IL8 and IL32]. At the same time, Tnf decreased the mRNA expression of anti-apoptotic genes (e.g. BCL2L1) and increased the mRNA expression of pro-apoptotic genes (e.g. BID). Overall, the results suggested that Tnf induced a complex set of pro-inflammatory and pro-apoptotic signals in stroma cells that promote apoptosis in malignant myeloid clones. Additional studies will be required to determine which of these signals are critical for the induction of apoptosis in the malignant clones. Those insights, in turn, may point the way to novel therapeutic approaches.

Notch1 Signaling Promotes Survival of Leukemic Cells in Close Contact with Bone Marrow-Derived Stromal Cells by Activating AKT and Stat-3 Pathways.

We have previously demonstrated that the bone marrow (BM) microenvironment plays a crucial role in the pathogenesis of acute myeloid leukemia (AML) by influencing tumor growth, survival, and drug resistance. Integrin-linked kinase (ILK) directly interacts with β integrins and phosphorylates AKT in a PI3-kinase(PI3K)-dependent manner. HES1 encodes a basic helix-loop-helix transcription factor downstream of the Notch receptor, and functions as a positive regulator of hematopoietic stem cell self-renewal. In this study, we investigated the functional role of Notch/HES1 signaling in leukemic cell survival stimulated by BM stromal interactions. Direct co-culture of human mesenchymal stem cell (MSC) and leukemic NB4 (AML) or REH (preB-ALL) cells activated ILK kinase activity and enhanced phosphorylation of AKT and GSK3β along with increased Notch1 and HES1 expression. Both, ILK inhibitor QLT0267 or PI3K inhibitor LY294002 inhibited MSC induced p-AKT, p-GSK3β and HES1 expression, while GSK3 inhibitor BIO induced HES1 expression, suggesting that activation of Notch signaling in stromal co-cultures is at least in part mediated via ILK/GSK3β pathway. Because the co-factor Mastermind-like (MAML) is required to transcribe downstream target genes of Notch pathway, we introduced a dominant-negative form of MAML to prevent Notch signaling. dnMAML blocked both basal and MSC-induced expression of cleaved intracellular Notch1 and HES1 and unexpectedly prevented MSC-induced phosphorylation of AktSer473, but not of GSK3βSer9, as documented by Western blot and confocal microscopy analyses (Figure 1A). Co-culture with MSC induced Stat3Tyr705 phosphorylation in NB4 cells, and this effect was abrogated by γ-secretase inhibitor (Fig. 1B). In contrast, Stat3 was still activated in leukemic cells harboring dnMAML (Fig. 1A), suggesting that Stat3Tyr705 phosphorylation is mediated via non-transcriptional effects of Notch or non-Notch effects of γ-secretase inhibition. Co-culture with MSC or with HS27A cells expressing Notch ligand Jagged1 stimulated REH cells proliferation under serum-limited conditions, which was partially blocked by γ-secretase inhibitor and completely abrogated in dnMAML REH cells. Interestingly, dnMAML NB4 cells acquired cell growth ability upon serum limitation.Altogether, these results suggest that interaction of leukemic and bone marrow stromal cells results in activation of PI3K/ILK/GSK3β signaling, the latter in turn activating Notch pathway. Notch activation enhances cell-type dependent leukemia cell survival upon interaction with BM-derived stromal cells. These effects of Notch signaling are at least in part mediated by a feedback activation of AKT pathway in a transcription-dependent manner, and via activation of Stat3 signaling independent of MAML. Our data show Notch-mediated regulation of AKT/Stat3 pathways and provide a novel role for activated Notch signaling in the context of bone marrow microenvironment. [Display omitted] [Display omitted]

Hypoxia increases VEGF-A production by prostate cancer and bone marrow stromal cells and initiates paracrine activation of bone marrow endothelial cells

Hypoxia develops at sites of rapid cancer growth near sites of poorly organized vasculature. Heparin binding growth factors (HBGFs) support neoangiogenesis of tumors. We examined the effect of culturing bone-targeted, metastatic C4-2B prostate cancer cells and bone stromal derived HS27a cells under hypoxic conditions on expression of vascular endothelial growth factor (VEGF) family members. A sealed chamber infused with 1% (hypoxic) or 20% (normoxic) O(2) was used. Both cell lines produced VEGF-A in normoxia, but little or no HB-EGF, another HBGF. HS27a cells produced low levels of FGF-2 and HGF, but little or none was secreted by C4-2B cells. Levels of VEGF-A in conditioned medium (CM) from both cell lines doubled when cultured in hypoxia. Similar changes in VEGF-A mRNA levels were seen. Receptor expression was unchanged by hypoxia. Changes in VEGF-A expression during hypoxia were preceded by nuclear accumulation of hypoxia inducible factor-1alpha (HIF-1alpha). Bone marrow endothelial (BME) cells express high levels of VEGFR2/flk-1, and are targets of VEGF-A induced neovascularization. BME cells proliferated in response to treatment with HS27a CM, but not C4-2B CM. BME cells formed tube-like angiogenic structures on growth factor reduced Matrigel in response to CM from HS27a or C4-2B cells. This response was greater when CM was produced under hypoxia, and was reduced by VEGF-A or FGF-2 neutralizing antibodies. We conclude that hypoxia triggers a physiologically relevant increase in VEGF-A by prostate cancer and bone marrow stromal cells which involves a paracrine loop that recruits and activates BME to support tumor neovascularization-related processes.

Co-Transplantation of Stromal Cells Enhances Engraftment Ability of MDS-Derived Hematopoietic Precursors in NOD/SCID-b2microglobulin-Deficient (b2mnull) Mice.

Propagation of hemopoietic clones derived from MDS marrow in vitro or in vivo has proven difficult. We recently showed engraftment of clonal cells identified by fluorescence in situ hybridization (FISH) markers, in b2mnull mice using MDS bone marrow mononuclear cells (BMMC) injected intramedullarly along with the human stroma-derived cell lines, HS5 and HS27a. To define the role of stromal cells for transplant success, we conducted additional in vitro and in vivo studies.Methods:Using NOD/SCID-b2mnull mice (irradiated with 350cGy), MDS BMMC (107cells) or purified CD34+ MDS-derived hematopoietic precursors (106cells) were transplanted into the right femur with or without the addition of HS5 and HS27a (2x105) stroma cells. Engraftment was defined as presence of ≥ 0.2% human CD45+ cells in peripheral blood.To document the presence of human stroma, mice were transplanted with GFP transduced HS5 and HS27a cells, and sacrificed 1h or 24h after transplant. Marrow cells from the femora were evaluated for the presence of GFP signals by flow cytometry.MDS-derived hematopoietic precursors from patients with identifiable FISH markers, were sorted from the blast gate (CD45 dim, low side scatter [ SSC]), and plated on stromal feeder layers (either HS5 or HS27a, irradiated with 1800cGy), in contact or transwell cultures. After one week, cells were recovered and analyzed by FISH, and also plated in methocult. Colonies were plucked at two weeks and also analyzed by FISH.Results: 11 of 13 mice transplanted without co-injection of stroma cells showed engraftment of human cells. At six weeks, clonal cells were detected in four of six mice with a FISH marker [−5 and del(5q)], suggesting that stromal cells were not an absolute requirement for the propagation of clonal cells. However, in mice transplanted with cells from the same marrow source with or without stromal cells, the engraftment rate was higher with stroma (4/7, 57%) than without stroma (2/6, 33%), as was the proportion of human hemopoietic cells in marrow (6.4±2.7% vs 2.2±0.4%, respectively). The proportion of human cells was higher at the injection site than in the contralateral femur. Furthermore, clonal (FISH+) cells were detected in 3 of 4 mice in the stroma group (del(5q) and +8), but not in two mice that were not injected with stroma but showed engraftment of “normal” human cells. Among mice transplanted with cells from a patient with +8, only those in the stroma group showed engraftment (clonal and non-clonal cells). Nine mice transplanted with CD34+ MDS cells (7 with and 2 without addition of stroma) did not show engraftment, suggesting that not only stroma but also other accessory cells are important for the tranplant success. GFP+ stromal cells were recognized in mouse bone marrow obtained from the injection site, but not contralaterally. In vitro, clonal cells (−7) were recovered from short-term assays on HS5 or HS27a cells, from contact and trans-well cultures. However, colonies were formed only from cells in contact cultures; HS5 stroma (a rich source of cytokines) favored clonal (FISH+) cell growth.Conclusions: Thus, the intramedullary co-transplantation of stromal cells enhanced the engraftment ability of normal and clonal cells from human MDS marrow. In vitro data suggest that cell-to-cell contact is necessary, and that clonal cell growth may have an advantage in the presence of HS5 cells.

BMP4 Induces Changes in Jagged 1 Expression in Bone Marrow Stroma: Association with Induction of Notch Regulated Genes in CD34+Cells.

In a previous report we identified gene products that may be associated with stem cell maintenance by comparative transcriptome analysis of 2 functionally distinct stromal cell lines: HS-27a which supports primitive hematopoietic progenitor cells and HS-5 which stimulates differentiation. Since the ability of stromal cells to maintain stem cells is lost as the percentage of monocytes in stromal cultures increases, monokine-induced changes in HS-27a gene expression were also determined. An algorithm that combined these datasets was developed and used to identify factors produced by stroma that could be hypothesized to influence hematopoietic stem cell fate. Bone Morphogenetic protein 4 (BMP4) was identified and selected for study. Real time quantitative PCR confirmed that BMP4 gene expression was 9 fold higher in HS-27a than HS-5 and suppressed 6-fold by IL-1β. BMP4 protein secretion followed a similar pattern: HS-27a cells secreted 70 pg/ml BMP4 protein and treatment with IL-1β resulted in a 3 fold suppression; no BMP-4 secretion was detected from HS-5 cells. BMP4 is a critical factor for regulating hematopoietic development during embryogenesis and is involved in the regulation of T-cell differentiation by thymic stroma. However, relatively little is known about the role of bone marrow stromal derived BMP4 in adult hematopoiesis. BMP4 has been implicated in Notch signaling in muscle development. Since the Notch pathway is a key determinant of stem cell fate in hematopoiesis and the Notch ligand, Jagged 1, is differentially expressed in HS-5 and HS-27a cells, we investigated the effect of BMP4 on stromal expression of Jagged 1. We exposed HS-5 cells to BMP4 and assayed for Jagged 1 expression by western blot analysis. BMP4 induced both expression and modification of Jagged 1 in HS-5 cells. To determine if changes in Jagged 1 expression altered signaling between stroma and CD34+ cells, we exposed HS-5 cells to BMP4 for 24 hours. The medium was subsequently removed and replaced with fresh medium that did not contain BMP4. CD34+ cells were then added to the HS-5 cells and incubated at 37° for 2 to 24 hours. CD34+ cells were collected for RNA extraction and whole cell protein extracts were made from the HS-5 cells to verify changes in Jagged 1 expression. Pre-incubation of HS-5 cells with BMP4 prior to co-culture with CD34+ cells resulted in a consistent increase (1.4 to 2.0 fold) in gene expression of the notch regulated genes, Hey1 and Hes1. Although other, as yet undefined, BMP4 induced changes in marrow stroma may be responsible for this induction, we hypothesize that BMP4-induced changes in stromal Jagged 1 expression increases Hey 1 and Hes 1 gene expression via ligand engagement and activation of Notch signaling. Taken together, these studies suggest that BMP4 acts indirectly on progenitor cells via bone marrow stroma through a previously undescribed mechanism whereby BMP4 induces changes in stromal cell expression of the Notch ligand, Jagged1.

Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein

DNA spotted microarrays were used to compare gene expression profiles from 2 functionally distinct human marrow stromal cell lines: HS-27a, which supports cobblestone area formation by early hematopoietic progenitors, and HS-5, which secretes multiple cytokines that support the proliferation of committed progenitors. One unexpected result was the high level of interleukin-7 receptor (IL-7R) gene expression in HS-27a stromal cells. Northern blot analysis confirmed the IL-7R RNA expression, and Western blots for the IL-7R protein detected both a full-length (90-kd) IL-7R and a smaller 30-kd fragment in both HS-27a cells and primary stromal cell cultures, whereas only the 90-kd receptor protein was detected in peripheral blood mononuclear cells. Biotinylated IL-7 was shown to bind to HS-27a cells under physiologic conditions, and this binding was inhibited by blocking anti-IL-7 antibodies. Tyrosine phosphorylation of several proteins (55 kd, 30 kd, and 24 kd) in HS-27a cells was rapidly increased after incubation with recombinant IL-7. One of the phosphorylated proteins proved to be the 30-kd IL-7R fragment. Exposure of HS-27a cells to IL-7 resulted in a 10-fold increase in secretion of IL-6 into culture supernatants but no increase in the cytokines stromal cell-derived factor 1, macrophage inflammatory protein 1 alpha, or IL-1 beta. The up-regulation of IL-6 secretion is associated with a rapid but transient increase in detectable levels of IL-6 messenger RNA. These data suggest that IL-7 may function to regulate the milieu of the microenvironment by modulating IL-6 secretion by the IL-7R-expressing stromal elements.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

AbstractA Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1α (IL-1α) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.