Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.
Cell surface glycans, such as heparan sulfate (HS), are increasingly identified as co-regulators of growth factor signaling in early embryonic development; therefore, chemical tailoring of HS activity within the cellular glycocalyx of stem cells offers an opportunity to control their differentiation. The growth factors FGF2 and BMP4 are involved in mediating the exit of murine embryonic stem cells (mESCs) from their pluripotent state and their differentiation toward mesodermal cell types, respectively. Here, we report a method for remodeling the glycocalyx of mutant Ext1-/- mESCs with defective biosynthesis of HS to drive their mesodermal differentiation in an embryoid body culture. Lipid-functionalized synthetic HS-mimetic glycopolymers with affinity for both FGF2 and BMP4 were introduced into the plasma membrane of Ext1-/- mESCs, where they acted as functional co-receptors of these growth factors and facilitated signal transduction through associated MAPK and Smad signaling pathways. We demonstrate that these materials can be employed to remodel Ext1-/- mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells.
Bone morphogenetic protein 4 (BMP4) has an important role in regulating cellular proliferation, differentiation and apoptosis. It, however, is still unclear as to the mechanisms by which BMP4 regulates the apoptosis of granulosa cells (GCs) in geese. In the present study, there was cloning of the full-length coding sequence of goose BMP4 gene, which consisted of 1212 nucleotides encoding 403 amino acids. Its deduced amino acid sequence comprised one signal peptide, one TGFβ pro-peptide and one mature peptide domain. Results from conducting the quantitative real-time PCR (qPCR) indicated the relative abundances of BMP4 mRNA in geese GCs increased gradually from the relative abundances in pre-hierarchical follicles that were 4 to 6 mm in diameter to that in the fifth largest (F5) follicle and then relative abundances of BMP4 mRNA decreased with further development as the largest (F1) follicle. Results from use of the TUNEL assay indicated that overexpression of the goose BMP4 gene suppressed GC apoptosis and this was confirmed when relative abundances of the CAD, Caspase-9 and Caspase-3 proteins were determined using western blotting. In addition, overexpression of the BMP4 gene induced phosphorylation of AKT, which was inhibited with use of the PI3K inhibitor, LY294002. Co-transfection of BMP4 and LY294002 resulted in increased relative abundances of Caspase-9 and CAD proteins but had no effect on that of Caspase-3. Taken together, these results suggested that expression of the BMP4 gene resulted in a reduction in Caspase-9 protein leading to inhibition of GC apoptosis via the PI3K/AKT signaling pathway in geese.
The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.
Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.
Urinary cell-free DNA (ucfDNA) has great potential as a "liquid biopsy" for use in diagnosis of urological cancers. In this study, we compared ucfDNA gene expression levels between patients with bladder cancer (BC) and those with hematuria, and determined whether they could be used as a noninvasive urine-based marker.
The study cohort of 355 patients included a screening group (40 BC and 41 hematuria controls) and a validation cohort (149 BC and 125 hematuria controls). Expression levels ratios of 1 up-regulated gene (IQGAP3) to those of 7 down-regulated genes were examined in ucfDNA in the screening group to identify ratios that differed significantly between BC and hematuria patients. IQGAP3/BMP4 and IQGAP3/FAM107A ratios were selected and combined to develop a discriminant score (DS) index, which was tested in the validation cohort. Receiver operating characteristic curves and areas under the curve were calculated to evaluate the performance of the DS index.
IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were both significantly higher in BC patients than in hematuria patients (both P < 0.001). The DS index had an area under the curve of 0.862, a sensitivity of 71.0%, a specificity of 88.6%, a positive predictive value of 90.3%, and a negative predictive value of 67.2%.
Both IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were significantly higher in patients with BC than in those with hematuria. The DS index exhibits good diagnostic performance as a noninvasive biomarker.
Many human gene mutations have been linked to congenital heart disease (CHD), yet CHD remains a major health issue worldwide due in part to an incomplete understanding of the molecular basis for cardiac malformation.
Here we identify the orthologous mouse Pou6f1 and zebrafish pouC as POU homeodomain transcription factors enriched in the developing heart. We find that pouC is a multi-functional transcriptional regulator containing separable activation, repression, protein-protein interaction, and DNA binding domains. Using zebrafish heart development as a model system, we demonstrate that pouC knockdown impairs cardiac morphogenesis and affects cardiovascular function. We also find that levels of pouC expression must be fine-tuned to enable proper heart formation. At the cellular level, we demonstrate that pouC knockdown disrupts atrioventricular canal (AVC) cardiomyocyte maintenance, although chamber myocyte specification remains intact. Mechanistically, we show that pouC binds a bmp4 intronic regulatory element to mediate transcriptional activation.
Taken together, our study establishes pouC as a novel transcriptional input into the regulatory hierarchy that drives AVC morphogenesis in zebrafish. We anticipate that these findings will inform future efforts to explore functional conservation in mammals and potential association with atrioventricular septal defects (AVSDs) in humans. This article is protected by copyright. All rights reserved.
Cancer-associated fibroblasts (CAFs) demonstrate the characteristics of myofibroblast differentiation by often expressing the ultrastructure of alpha-smooth muscle actin (αSMA). However, heterogeneity among cancer-associated fibroblasts (CAFs), with respect to αSMA expression, has been demonstrated in several clinical studies of oral cancer. Like normal stem cells, stem-like cancer cells (SLCCs) are also regulated extrinsically by its microenvironment; therefore, we postulated that the heterogeneous oral-CAFs would differently regulate oral-SLCCs. Using transcriptomics, we clearly demonstrated that the gene expression differences between oral tumor-derived CAFs were indeed the molecular basis of heterogeneity. This also grouped these CAFs in two distinct clusters, which were named as C1 and C2. Interestingly, the oral-CAFs belonging to C1 or C2 clusters showed low or high αSMA-score, respectively. Our data with tumor tissues and in vitro co-culture experiments interestingly demonstrated a negative correlation between αSMA-score and cell proliferation, whereas, the frequency of oral-SLCCs was significantly positively correlated with αSMA-score. The oral-CAF-subtype with lower score for αSMA (C1-type CAFs) was more supportive for cell proliferation but suppressive for the self-renewal growth of oral-SLCCs. Further, we found the determining role of BMP4 in C1-type CAFs-mediated suppression of self-renewal of oral-SLCCs. Overall, we have discovered an unexplored interaction between CAFs with lower-αSMA expression and SLCCs in oral tumors and provided the first evidence about the involvement of CAF-expressed BMP4 in regulation of self-renewal of oral-SLCCs.
Lung cancer is the leading cause of cancer-associated deaths worldwide. In particular, non-small-cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR) mutations are associated with resistance development of EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment. Recent findings suggest that bone morphogenetic proteins (BMPs) and microRNAs (miRNAs) might act as oncogenes or tumor suppressors in the tumor microenvironment. In this study, for the first time, we identified the potential roles of BMPs and miRNAs involved in EGFR-TKI resistance by analyzing datasets from a pair of parental cells and NSCLC cells with acquired EGFR-TKI resistance. BMP4 was observed to be significantly overexpressed in the EGFR-TKI-resistant cells, and its mechanism of action was strongly associated with the induction of cancer cell energy metabolism through the modulation of Acyl-CoA synthetase long-chain family member 4. In addition, miR-139-5p was observed to be significantly downregulated in the resistant NSCLC cells. The combination of miR-139-5p and yuanhuadine, a naturally derived antitumor agent, synergistically suppressed BMP4 expression in the resistant cells. We further confirmed that LDN-193189, a small molecule BMP receptor 1 inhibitor, effectively inhibited tumor growth in a xenograft nude mouse model implanted with the EFGR-TKI-resistant cells. These findings suggest a novel role of BMP4-mediated tumorigenesis in the progression of acquired drug resistance in EGFR-mutant NSCLC cells.
BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.