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In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
Copyright © 2017 Elsevier Inc. All rights reserved.
Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings.
Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Transcription of BMPs and their antagonists in precise spatiotemporal patterns is essential for proper skeletal development, maturation, maintenance, and repair. Nevertheless, transcriptional activity of these molecules in skeletal tissues beyond embryogenesis has not been well characterized. In this study, we used several transgenic reporter mouse lines to define the transcriptional activity of two potent BMP ligands, Bmp2 and Bmp4, and their antagonist, Noggin, in the postnatal skeleton. At 3 to 4 weeks of age, Bmp4 and Noggin reporter activity was readily apparent in most cells of the osteogenic or chondrogenic lineages, respectively, whereas Bmp2 reporter activity was strongest in terminally differentiated cells of both lineages. By 5 to 6 months, activity of the reporters had generally abated; however, the Noggin and Bmp2 reporters remained remarkably active in articular chondrocytes and persisted there indefinitely. We further found that endogenous Bmp2, Bmp4, and Noggin transcript levels in postnatal bone and cartilage mirrored the activity of their respective reporters in these tissues. Finally, we found that the activity of the Bmp2, Bmp4, and Noggin reporters in bone and cartilage at 3 to 4 weeks could be recapitulated in both osteogenic and chondrogenic culture models. These results reveal that Bmp2, Bmp4, and Noggin transcription persists to varying degrees in skeletal tissues postnatally, with each gene exhibiting its own cell type-specific pattern of activity. Illuminating these patterns and their dynamics will guide future studies aimed at elucidating both the causes and consequences of aberrant BMP signaling in the postnatal skeleton.
© 2014 American Society for Bone and Mineral Research.
Bone Morphogenetic Proteins (BMPs) are secreted cytokines that are part of the Transforming Growth Factor β (TGFβ) superfamily. BMPs have been shown to be highly expressed in human breast cancers, and loss of BMP signaling in mammary carcinomas has been shown to accelerate metastases. Interestingly, other work has indicated that stimulation of dermal fibroblasts with BMP can enhance secretion of pro-tumorigenic factors. Furthermore, treatment of carcinoma-associated fibroblasts (CAFs) derived from a mouse prostate carcinoma with BMP4 was shown to stimulate angiogenesis. We sought to determine the effect of BMP treatment on mammary fibroblasts. A large number of secreted pro-inflammatory cytokines and matrix-metallo proteases (MMPs) were found to be upregulated in response to BMP4 treatment. Fibroblasts that were stimulated with BMP4 were found to enhance mammary carcinoma cell invasion, and these effects were inhibited by a BMP receptor kinase antagonist. Treatment with BMP in turn elevated pro-tumorigenic secreted factors such as IL-6 and MMP-3. These experiments demonstrate that BMP may stimulate tumor progression within the tumor microenvironment.
Interferon regulatory factor 6 (IRF6) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in IRF6 lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the Myf5+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6-/- embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.
PURPOSE - Pax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity.
METHODS - We conditionally deleted Fgfr2 in ST (Fgfr2(ST-/-)) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects.
RESULTS - We confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2(ST-/-) mice at embryonic day (E) 10.5. E11.5 Fgfr2(ST-/-) mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2(ST-/-) mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2(ST-/-) mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes.
CONCLUSION - Mutations in FGFR2 could possibly cause VUR in humans.
Transforming growth factor (TGF)-β family members exert strong effects on restoration of liver mass after injury. Bone morphogenetic proteins (BMPs) are members of the TGF-β family and are found in the liver, suggesting that these proteins may play a role in liver regeneration. We examined BMP signaling in the liver during hepatectomy. We found that BMP4 is constitutively expressed in the peribiliary stroma and endothelial cells of the liver and that expression is decreased after hepatectomy. Mice driven to maintain BMP4 expression in the liver display inhibited hepatocyte proliferation and restoration of liver mass after hepatectomy, suggesting that reduced BMP4 is necessary for normal regeneration. Consistent with this finding, hepatocyte-specific deletion of the BMP receptor activin receptor-like kinase 3 (Alk3) enhances regeneration and reduces phosphorylation of SMAD1/5/8, a transducer of BMP signaling. In contrast to experiments in wild-type mice, maintaining BMP4 levels has no effect on liver regeneration in hepatocyte-specific Alk3 null mice, providing evidence that BMP4 signals through Alk3 to inhibit liver regeneration. Consistent with these findings, the BMP4 antagonist Noggin enhances regeneration. Furthermore, high-dose BMP4 inhibits proliferation of primary hepatocytes and HepG2 cells in culture. These findings elucidate a new, potentially clinically relevant paradigm in which a constitutively expressed paracrine inhibitory factor plays a critical role in liver regeneration.
During renal development, the proper emergence of the ureteric bud (UB) from the Wolffian duct is essential for formation of the urinary system. Previously, we showed that expression of transcription factor GATA-2 in the urogenital primordium was demarcated anteroposteriorly into two domains that were regulated by separate enhancers. While GATA-2 expression in the caudal urogenital mesenchyme is controlled by the UG4 enhancer, its more-rostral expression is regulated by UG2. We found that anteriorly displaced budding led to obstructed megaureters in Gata2 hypomorphic mutant mice, possibly due to reduced expression of the downstream effector bone morphogenetic protein 4 (BMP4). Here, we report that UG4-driven, but not UG2-driven, GATA-2 expression in the urogenital mesenchyme significantly reverts the uropathy observed in the Gata2 hypomorphic mutant mice. Furthermore, the data show that transgenic rescue by GATA-2 reverses the rostral outgrowth of the UB. We also provide evidence for a GATA-2-BMP4 epistatic relationship by demonstrating that reporter gene expression from a Bmp4 bacterial artificial chromosome (BAC) transgene is altered in Gata2 hypomorphs; furthermore, UG4-directed BMP4 expression in the mutants leads to reduced incidence of megaureters. These results demonstrate that GATA-2 expression in the caudal urogenital mesenchyme as directed by the UG4 enhancer is crucial for proper development of the urinary tract and that its regulation of BMP4 expression is a critical aspect of this function.
Maldevelopment of outflow tract and aortic arch arteries is among the most common forms of human congenital heart diseases. Both Bmp4 and Tbx1 are known to play critical roles during cardiovascular development. Expression of these two genes partially overlaps in pharyngeal arch areas in mouse embryos. In this study, we applied a conditional gene inactivation approach to test the hypothesis that Bmp4 expressed from the Tbx1 expression domain plays a critical role for normal development of outflow tract and pharyngeal arch arteries. We showed that inactivation of Bmp4 from Tbx1-expressing cells leads to the spectrum of deformities resembling the cardiovascular defects observed in human DiGeorge syndrome patients. Inactivation of Bmp4 from the Tbx1 expression domain did not cause patterning defects, but affected remodeling of outflow tract and pharyngeal arch arteries. Our further examination revealed that Bmp4 is required for normal recruitment/differentiation of smooth muscle cells surrounding the PAA4 and survival of outflow tract cushion mesenchymal cells.
Copyright © 2010 S. Karger AG, Basel.
The bone morphogenetic protein (BMP) type 2 receptor ligand, Bmp2, is upregulated in the peripheral pulmonary vasculature during hypoxia-induced pulmonary hypertension (PH). This contrasts with the expression of Bmp4, which is expressed in respiratory epithelia throughout the lung. Unlike heterozygous null Bmp4 mice (Bmp4(LacZ/+)), which are protected from the development of hypoxic PH, mice that are heterozygous null for Bmp2 (Bmp2(+/-)) develop more severe hypoxic PH than their wild-type littermates. This is associated with reduced endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2(+/-) but not Bmp4(LacZ/+) mutant mice. Furthermore, exogenous BMP2 upregulates eNOS expression and activity in intrapulmonary artery and pulmonary endothelial cell preparations, indicating that eNOS is a target of Bmp2 signaling in the pulmonary vasculature. Together, these data demonstrate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH and suggest that the protective effects of Bmp2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature.