Role Of BMP4 Research Articles

Mechanisms Governing Endogenous Thymic Regeneration

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in their recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also contribute to thymic repair. Unbiased transcriptome analysis on the damage-resistant non-hematopoietic compartemtn of the thymus revealed significant upregulation of Bmp4 and its downstream signalling targets (Fig. 1a). Further interrogation revealed that while thymic expression of BMP4 was restricted to fibroblasts and endothelial cells (ECs), only ECs increase their expression of Bmp4 after damage; and specific and inducible deletion of BMP4 in ECs led to significantly worse regeneration (Fig. 1b).Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration has thus far remained largely unclear. Careful interrogation of ECs after damage revealed that, much like ILCs, ECs are extremely resistant to multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and TBI. However, whole organ imaging analysis using light sheet field microscopy suggested that even though the number of ECs remain unchanged after damage, there is considerable structural changes to the vasculature including shortening of the vessels and reduced branching. Although BMP4 receptors are widely expressed in the thymus, there was enriched expression for BMP4 receptor subunits on TECs, which is consistent with the role of BMP4 in thymus ontogeny by promoting TEC development, at least partially due to its ability to induce expression of Foxn1 (Fig. 1c), a key transcription factor for the development and maintenance of TECs. Consistent with these findings, after thymic damage we observed a significant increase in the expression of Foxn1 after damage as well as GSEA enrichment for downstream FOXN1 target genes (Fig. 1d); including Dll4, the Notch ligand critical for T cell development and whose concentration we have previously shown can directly regulate thymic size (Velardi 2014 J Exp Med 211:2341). Finally, using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function; but only when the exEC were derived from the thymus but not from heart or kidney (Fig. 1e). Consistent with endogenous regeneration, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 and Dll4 .Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic renewal. These studies thus not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. [Display omitted] Disclosuresvan den Brink:PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy.

Bone morphogenetic protein 7-transduced human dermal-derived fibroblast cells differentiate into osteoblasts and form bone in vivo

ABSTRACTBackground: Human dermal-derived fibroblast cells (hDDFCs) are multipotent. Bone morphogenetic proteins (BMPs) are a group of cytokines that promote different developmental processes, including the formation of bone. BMPs can promote hDDFC osteogenesis, but the role of BMP7 in hDDFC osteogenesis in vitro and bone formation in vivo has not been investigated in depth. Materials and Methods: hDDFCs were stably transfected with a human BMP7 recombinant adenovirus and osteogenic differentiation was examined by alkaline phosphatase staining and calcium accumulation. In addition, we measured the expression of osteoblast-related genes. To examine osteogenesis in vivo, we injected C57BL/6 nude mice with adenovirus-transfected hDDFCs in a calcium alginate hydrogel and examined bone formation using soft X-ray, histological, and immunohistochemical analyses. Results: Our findings showed that adenovirus-mediated BMP7 expression promoted osteogenic differentiation of hDDFCs and enhanced expression of osteoblast-related genes in vitro. Cells infected with BMP7 adenoviruses showed enhanced bone formation and osteoblast-related gene expression in vivo after the injection of hDDFC–hydrogel mixture. Conclusions: Taken together, our data indicate that BMP7 significantly promotes hDDFC osteogenesis, and confirm that infecting hDDFCs with BMP7-expressing adenoviruses is a useful tool for bone tissue engineering.

Inhibitory Role Of BMP4 On The PDGF‐Induced Proliferation And Collagen Synthesis In PASMCs

Pulmonary arterial hypertension (PAH) is a severe lung disease characterized by remodeling of pulmonary vascular walls due to the abnormal vascular smooth muscle cell (VSMC) proliferation and accumulation of extracellular matrix proteins such as collagen. Our recent work shows that platelet‐derived growth factor‐BB (PDGF‐BB) induces cell proliferation and collagen synthesis in pulmonary artery SMC (PASMCs) via extracellular signal–regulated kinases (ERK)‐dependent calpain activation in PAH. The aim of the present study to determine the effect of bone morphogenetic protein 4 (BMP4) on PDGF‐stimulated calpain‐Transforming growth factor beta 1 (TGFβ1) signaling in PASMCs. PASMCs from normal and PAH patients were incubated with PDGF‐BB in the presence and absence of BMP4 for 0.5 to 24 h after which protein levels of collagen‐I, p‐Smad2/3, p‐Smad1/5, p‐ERK and intracellular active TGFβ1, calpain activity and cell proliferation were measured. We found that BMP4 augmented the protein levels of p‐Smad1/5 but did not influence the protein contents of collagen‐I, p‐Smad2/3, p‐ERK, active TGFβ1, and calpain activity in normal PASMCs. However, BMP4 attenuated the PDGF‐induced increases in calpain activity, cell proliferation and protein levels of collagen‐I, p‐Smad2/3, and active TGFβ1 in control cells. The results also showed that BMP4 did not alter the PDGF‐induced increases in p‐ERK or p‐Src. In addition, downregulation of Smad1/5 did not prevent the negative effects of BMP4 on the PDGF‐activated signal pathway. Furthermore, BMP4 increased the PKA activity and blocked the PDGF‐induced decrease in calpain‐2 phosphorylation at Ser369 in normal PASMCs. Inhibition of PKA prevented the inhibitory effects of BMP4 on calpain activation and dephosphorylation of Ser369 induced by PDGF. In addition, PKA activator forskolin recapitulated the inhibitory effect of BMP4 on PDGF‐stimulated calpain activation in normal PASMCs. In contrast, the suppressive effects of BMP4 on PDGF‐induced cell proliferation and collagen synthesis were abrogated in PASMCs from PAH patients. BMP4‐evoked PKA activation was diminished in PASMCs from PAH patients and BMP4 was not able to prevent the PDGF‐induced reduction in S369 phosphorylation and activation of calpain. Collectively, these data demonstrate that BMP4 inhibits PDGF‐induced cell proliferation and collagen synthesis via PKA‐mediated inhibition of calpain‐2 in normal PASMCs. Impairment of the BMP4‐PKA signaling may contribute to elevated calpain activation and pulmonary vascular remodeling in PAH.Support or Funding InformationSupported by FAMRI CIA140083 (YS), Department of Veterans Affairs BX002035 (YS) and AHA 16POST27730023 (LK).

BMP7 Induces Uterine Receptivity and Blastocyst Attachment.

In women, the window of implantation is limited to a brief 2- to 3-day period characterized by optimal levels of circulating ovarian hormones and a receptive endometrium. Although the window of implantation is assumed to occur 8 to 10 days after ovulation in women, molecular markers of endometrial receptivity are necessary to determine optimal timing prior to embryo transfer. Previous studies showed that members of the bone morphogenetic protein (BMP) family are expressed in the uterus necessary for female fertility; however, the role of BMP7 during implantation and in late gestation is not known. To determine the contribution of BMP7 to female fertility, we generated Bmp7flox/flox-Pgr-cre+/- [BMP7 conditional knockout (cKO)] mice. We found that absence of BMP7 in the female reproductive tract resulted in subfertility due to uterine defects. At the time of implantation, BMP7 cKO females displayed a nonreceptive endometrium with elevated estrogen-dependent signaling. These implantation-related defects also affected decidualization and resulted in decreased expression of decidual cell markers such as Wnt4, Cox2, Ereg, and Bmp2. We also observed placental abnormalities in pregnant Bmp7 cKO mice, including excessive parietal trophoblast giant cells and absence of a mature placenta at 10.5 days post coitum. To establish possible redundant roles of BMP5 and BMP7 during pregnancy, we generated double BMP5 knockout/BMP7 cKO [BMP5/7 double knockout (DKO)] mice; however, we found that the combined deletion had no additive disruptive effect on fertility. Our studies indicate that BMP7 is an important factor during the process of implantation that contributes to healthy embryonic development.

Upper limb non-unions treated with BMP-7: efficacy and clinical results

The management of upper limb non-unions can be challenging and often with unpredictable outcomes. In this study we present our experience with the use of BMP-7 in the treatment of upper limb non-unions.Between 2004 and 2011 all consecutive patients who were treated with BMP-7 were followed up prospectively until fracture union. Fracture union was assessed with regular radiological and clinical assessment. At the final follow up clinical assessment included the short Disabilities of the Arm, Shoulder and Hand (DASH) score. The minimum follow up was 12 months (12–36).In total 42 patients met the inclusion criteria with a mean age of 47 years. Anatomical distribution of the nonunion sites included 19 cases of mid/proximal forearm, 14 humeri, 6 distal radius and 3 clavicle. 35 patients had atrophic non-union, 11 had previous open fractures, and 10 had bone loss (range 1–3 cm). The mean number of operations performed and the mean time from injury to BMP-7 application was 1.5 and 26 months, respectively. 40 fractures had both clinical and radiological union whereas 2 patients had partial radiological union but a pain free range of motion. BMP-7 was applied in isolation in 1 case and in 41 cases the application was combined with autologous bone grafting. DASH scores were available at final follow up in 23 (55%) patients with a mean of 33 score (range 2–86.4).This study supports the view that the combination of ABG and BMP-7 can be considered as a successful treatment modality for the treatment of recalcitrant upper limb non-unions. Further studies preferably randomised controlled trials are desirable to throw more light into the role of BMP-7 in the treatment of upper limb nonunions.

A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells

BackgroundBone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AimsTo assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. MethodsChemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of β2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. ResultsHigh levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of β2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. ConclusionBMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of β2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases

The gene encoding bone morphogenetic protein-7 (Bmp7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of Bmp7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of Bmp7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.

Conserved and divergent expression patterns of markers of axial development in eutherian mammals.

Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.

Inflammatory Cytokines Stimulate Bone Morphogenetic Protein-2 Expression and Release from Pancreatic Beta Cells.

The proinflammatory cytokines interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ) play important roles in the progressive loss of beta-cell mass and function during development of both type 1 and type 2 diabetes. We have recently showed that bone morphogenetic protein (BMP)-2 and -4 are expressed in pancreatic islets and inhibit beta-cell growth and function. In this study, we describe that IL-1β and IFN-γ induce the expression of BMP-2 suggesting a possible role for BMP-2 in mediating the effects of IL-1β and IFN-γ on beta-cell apoptosis and dysfunction. IL-1β increased BMP-2 mRNA levels 6- and 3-fold in isolated islets of Langerhans from neonatal rat and human. Downstream target genes of the BMP pathway were also increased by cytokine treatment and could be reversed by neutralization of endogenous BMP activity. Nuclear factor kappa B- (NFκB) binding sites were identified in the rat BMP-2 promoter, and reporter assays verified the role of NFκB in cytokine-induced BMP-2 expression. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed NFκB binding to BMP-2 promoter upon IL-1β stimulation in beta cells. In conclusion, we suggest that NFκB stimulates BMP-2 mRNA expression in rat and human beta cells upon cytokine exposure.

Bone Morphogenic Protein 4 Mediates NOX1-Dependent eNOS Uncoupling, Endothelial Dysfunction, and COX2 Induction in Type 2 Diabetes Mellitus.

We have recently shown that angiotensin II-mediated uncoupling of endothelial nitric oxide synthase (eNOS) contributes to endothelial dysfunction in streptozotocin-induced type 1 diabetes mellitus. However, it has remained unclear whether and how eNOS uncoupling occurs in type 2 diabetes mellitus (T2DM) and the consequences of such in regulating vascular function. Here we investigated a role of bone morphogenic protein (BMP)-4 in mediating eNOS uncoupling, endothelial dysfunction, and inflammation in db/db mice. Circulating levels of BMP4 were markedly elevated in db/db mice but not in mice with type 1 diabetes mellitus, in which angiotensin II levels were significantly increased. Infusion of BMP4 antagonist noggin into db/db mice (15 μg/kg/day, 4 weeks) abolished eNOS uncoupling activity while restoring tetrahydrobiopterin (H(4)B) bioavailability. The impaired endothelium-dependent vasorelaxation in db/db aortas was significantly improved by noggin infusion. Exposure of aortic endothelial cells to BMP4 (50 ng/mL, 24 hours) resulted in eNOS uncoupling, which was attenuated by H(4)B precursor sepiapterin or small interfering RNA silencing nicotinamide adenine dinucleotide phosphate oxidase isoform 1 (NOX1). Interestingly, BMP4-dependent NOX1 up-regulation was abrogated by sepiapterin, implicating a NOX1-uncoupled eNOS-NOX1 feed-forward loop. BMP4 induction of cyclooxygenase 2 (COX2) expression and vascular cell adhesion protein 1 was found in db/db mice. Consistently, COX2 was up-regulated by BMP4 in endothelial cells, which was attenuated by sepiapterin, implicating an upstream role of eNOS uncoupling in COX2-mediated inflammatory activation. Taken together, our data for the first time reveal a novel role of BMP4 in inducing NOX1-dependent eNOS uncoupling in T2DM, which may promote development of novel therapeutics restoring endothelial function in T2DM.

The role of BMP2 in the pathophysiology of obesity

The role of BMP2 in the pathophysiology of obesity

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Role of BMP2 in the maturation and maintenance of the knee joint

Role of BMP2 in the maturation and maintenance of the knee joint

Local expression and role of BMP-2/4 in injured spinal cord.

We investigated local changes in BMP-2/4 expression in rat spinal cords 1 week following injury to study the damage effects of BMP-2/4 in spinal cord injury (SCI). Sprague Dawley rats (45, 4 months old) were randomized into three groups comprising 15 rats each: a SHAM group, an SCI without noggin group (SCIO), and an SCI with noggin group (SCID). The SCIO and SCID groups were subjected to spinal cord hemisection, and motor activity was assessed using the BBB score. Expression of BMP-2/4 in each injured spinal cord section was examined by hematoxylin and eosin staining, immunohistochemistry, and western blot. There were no significant differences in BBB scores among the three groups (P > 0.05). Following hemisection, the BBB score in the SHAM group was significantly higher than in the other two groups on the 1st day after modeling (P < 0.05), and the BBB scores in the SCIO and SCID groups were not significantly different (P > 0.05). Seven days after modeling, the BBB score in the SHAM group was significantly higher than in the other two groups (P < 0.05), and the BBB score in the SCID group was obviously higher than in the SCIO group (P < 0.05). The expression of BMP-2/4 was highest in the SCIO group and lowest in the SHAM group (P < 0.05). SCI can cause severe impairment of motor activity in rats. Seven days after SCI, the local expression of BMP-2/4 had obviously increased; noggin can effectively inhibit the expression of BMP-2/4 and reduce impairment.

Osteoblast maturation on microtextured titanium involves paracrine regulation of bone morphogenetic protein signaling.

Osteoblasts are sensitive to surface microtopography and chemistry. Osteoblast differentiation and maturation are higher in vitro and bone formation and osseointegration enhanced in vivo on microstructured titanium (Ti) compared to smooth surfaces. Cells increased BMP2 expression on microtextured Ti alloy, suggesting a paracrine role in regulating osteoblast maturation. However, recent studies show that exogenous BMP2 inhibits osteoblast production of anti-inflammatory cytokines and osteocalcin, indicating that control of BMP-signaling may be involved. This study examined whether cells modulate BMP ligands, receptors, and inhibitors during osteoblast maturation on Ti, specifically focusing on the roles of BMP2 and Noggin (NOG). mRNA and protein for BMP2, BMP4, and BMP7 and receptors BMPR1A, BMPR1B, and BMPR2, and BMP inhibitors were upregulated on microtextured surfaces in comparison to smooth surfaces. Maturation on microstructured Ti was slightly enhanced with exogenous BMP2 while NOG addition inhibited osteoblast maturation. Cells with NOG knocked down significantly increased osteoblast maturation. These results demonstrate that BMP-related molecules are controlled during osteoblast maturation on microstructured Ti surfaces and that endogenous NOG is an important regulator of the process. Modifying paracrine BMP signaling may yield more robust bone formation than application of exogenous BMPs.

Abstract P5-03-15: Epithelial-to-mesenchymal transition; what is the impact on breast cancer stem cells and drug resistance

Abstract Introduction: SP cells are a subpopulation of Cancer Stem Cells (CSCs) which have been isolated from many types of tissue, including the breast. SP cells can be sorted due to their ability to expel Hoechst 33342 dye through members of ATP Binding Cassette (ABC) transporters superfamily of proteins. This property could explain their ability to also export cytotoxic drugs leading to drug resistant tumors. Epithelial-to-Mesenchymal Transition (EMT) is a process known to contribute to metastasis in cancer via the TGF-β signalling pathway and it mainly results in the downregulation of E-cadherin. Conversely, BMP-7 is a known inducer of the reverse process (MET), leading to inhibition of invasiveness and metastasis. TGF-β1 treatment in the breast cancer cell line MCF-7 eliminated the SP population, decreased the levels of ABCG2 expression and reduced cell viability in the presence of mitoxantrone. The role of BMP-7 on SP cells has not been explored. In this study, we investigated the effects of TGF-β1 treatment on SP cells of the MDA-MB-231 cell line. Autocrine production of TGF-β in the MDA-MB-231 cells is thought to be essential for their growth and maintenance. Therefore, we examined the effects of exogenous TGF-β1 and BMP-7 treatment on the SP cells. Methods: We have utilised FACS combined with Hoechst 33342 dye efflux to identify cells with an SP phenotype in the MDA-MB-231 cell line. Moreover, using standard techniques, including Real-Time PCR and ICC we determined the expression of TGF-β receptor genes (TGFB-RI and TGFB-RII) and the activation of the TGF-β signalling pathway (Smad 2/3 phosphorylation) when TGF-β1(5 ng/ml) is added to MDA-MB-231 cell cultures. Cultures were also treated with BMP-7 (100 ng/ml) and the impact on p-smad 1/5/8 was examined by ICC. Finally, we treated the total cell population with TGF-β1 and BMP-7 for 3 days and performed SP analysis. Results: The MDA-MB-231 cell line contains an SP population and it expresses receptors for TGF-β1. Successful activation of the corresponding signaling pathways is achieved as confirmed by the detection of p-smad 2/3 and p-smad 1/5/8 after TGF-β1 and BMP-7 treatment, respectively. TGF-β1 led to the depletion of the SP cells, whereas these effects were reversed upon the addition of the TGFB-RI antagonist, SB-505124. Interestingly, treatment with SB-505124 in the absence of TGF-β1 significantly increased the percentage of SP cells (p = 0.0285), but it also inhibited the cell growth of the total population. BMP-7 enhanced the SP numbers and this was confirmed by the use of the BMP-RI inhibitor, LDN193189. Conclusions: The presence of TGF-β1 leads to loss of the SP cell phenotype in the MDA-MB-231 cells, while BMP-7 has the opposite role. Definition of the exact EMT and MET mechanisms that regulate this subpopulation could enable the identification of new potential therapeutic targets and the development of more efficient strategies for the management of metastasis and drug resistance in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-15.

BMP4 and BMP7 induce the white-to-brown transition of primary human adipose stem cells

While white adipose tissue (AT) is an energy storage depot, brown AT is specialized in energy dissipation. Uncoupling protein 1 (UCP1)-expressing adipocytes with a different origin than classical brown adipocytes have been found in white AT. These "brite" (brown-in-white) adipocytes may represent a therapeutic target to counteract obesity. Bone morphogenetic proteins (BMPs) play a role in the regulation of adipogenesis. Based on studies with murine cells, BMP4 is assumed to induce stem cell commitment to the white adipocyte lineage, whereas BMP7 promotes brown adipogenesis. There is evidence for discrepancies between mouse and human AT. Therefore, we compared the effect of BMP4 and BMP7 on white-to-brown transition in primary human adipose stem cells (hASCs) from subcutaneous AT. Long-term exposure of hASCs to recombinant BMP4 or BMP7 during differentiation increased adipogenesis, as determined by lipid accumulation and peroxisome proliferator-activated receptor-γ (PPARγ) expression. Not only BMP7, but also BMP4, increased UCP1 expression in hASCs and decreased expression of the white-specific marker TCF21. The ability of hASCs to induce UCP1 in response to BMP4 and BMP7 markedly differed between donors and could be related to the expression of the brite marker CD137. However, mitochondrial content and oxygen consumption were not increased in hASCs challenged with BMP4 and BMP7. In conclusion, we showed for the first time that BMP4 has similar effects on white-to-brown transition as BMP7 in our human cell model. Thus the roles of BMP4 and BMP7 in adipogenesis cannot always be extrapolated from murine to human cell models.

Inhibition of BMP activity protects epithelial barrier function in lung injury

Epithelial injury is a central finding in pulmonary disease and is accompanied by disruption of epithelial barrier function, leading to pulmonary oedema and inflammation. Injured epithelial cells lose their properties and gain mesenchymal characteristics, a phenotypic switch that contributes to lung remodelling after injury. Here we studied bone morphogenetic protein (BMP) signalling and, in particular, the role of BMP2 and the BMP modulator BMPER in injured lung epithelium. Increased BMP activity, reflected by up-regulation of the Smad1/5-Id1 axis, is detected after injury of lung epithelium in vitro and in vivo. Two members of the BMP family, BMP2 and BMPER, have opposing effects. BMP2 is up-regulated after epithelial injury and causes epithelial dysfunction and hyperpermeability, mediated by the Smad1/5-Id1-dependent down-regulation of E-cadherin. In contrast, BMPER expression is decreased following injury, which in turn impairs epithelial integrity, characterized by reduction of E-cadherin and epithelial leakage in vitro and in vivo. High levels of BMPER antagonized BMP2-Smad5-Id1 signalling and prevented BMP2-mediated decrease of E-cadherin and hyperpermeability, suggesting that BMPER restores epithelial homeostasis. Supporting this notion, pharmacological inhibition of BMP signalling by LDN193189 prevented reduction of E-cadherin and disruption of epithelial barrier function. Inhibition of excessive BMP activation could be a new approach to restore epithelial integrity and prevent disruption of epithelial barrier function after lung injury.

BMP7 Expression in Esophageal Squamous Cell Carcinoma and Its Potential Role in Modulating Metastasis

Our previous study showed that BMP7 revealed significantly higher levels in esophageal squamous cell carcinoma (ESCC) tissues with lymph node metastasis compared with non-lymph node metastasis, using gene expression profiling assays. The roles of BMP7 in ESCC is not fully understood. The aim of this study was to investigate the effect of BMP7 on lymph node metastasis of ESCC and to explore its potential mechanism. Expression of BMP7 in ESCC tissues was evaluated by immunohistochemistry. BMP7 were down-regulated by RNA interference. The protein and mRNA levels of BMP7 were detected by western blot and RT-PCR, respectively. High content screening and transwell assay were used to identify the metastatic ability of tumor cells. Positivity of BMP7 staining was 57.5 % in the tissues of primary carcinoma with lymph node metastasis compared to tissues without lymph node metastasis, and expression of BMP7 was significantly higher in the cell lines with highly metastatic capacity than that in the cell lines without metastatic ability. Suppression of endogenous BMP7 expression by siRNA in the highly metastatic cell lines resulted in significant reduction in ability of cell migration and invasion in both in vitro and in vivo studies. In addition, inhibition of BMP7 by siRNA also leads to up-regulation of E-cadherin and down-regulation of MMP-9 in the highly metastatic cell lines. These findings indicate that BMP7 modulates the expression of E-cadherin and MMP-9, and by which mechanism it may regulate cell migration and metastasis of ESCCs.

A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of integrin-α4 expression

AbstractAlthough it is well established that BMP4 plays an important role in the development of hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis and how. Here, we describe a novel mechanism by which BMP4 regulates homing of murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment of murine BM derived c-kit+Lin−Sca-1+ (KLS) and CD150+CD48−KLS cells for up to 5 days in vitro prevented the culture-induced loss of Integrin-α4 (ITGA4) expression as well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmia-associated transcription factor (MITF), known to induce ITGA4 expression. Elevated ITGA4 expression significantly enhanced HSPC attachment to bone marrow stromal cells, homing and long-term engraftment of the BMP4 treated cells compared with the cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM derived Lin−CD34+ cells in culture, which was associated with improved homingpotential. Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution.