Bone morphogenetic protein 4 (BMP4)

Expression and distribution of bone morphogenetic protein 4 and its antagonist Noggin in the skin of Kazakh sheep (Ovis aries) with a white and brown coat color.

The natural coat color is an important trait of vertebrate animals. For example, the coat color can help avoid harm to human beings caused by chemical dyeing, and it has economic significance for domestic animals. The bone morphogenetic protein 4 (BMP4) and its antagonist Noggin can regulate pigmentation and the generation of coat color in mice; thus, they may also regulate the coat color of Kazakh sheep. To gain mechanistic insight into this possibility, we determined the relative expression levels of BMP4 and Noggin in the skin of white and brown Kazakh sheep by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis.

BMP-4

The localization of BMP4 and Noggin were detected by immunohistochemistry (IHC). The results of qPCR and western blot analysis demonstrated that the relative expression levels of BMP4 and Noggin in the skin of brown Kazakh sheep were significantly higher than those in white Kazakh sheep. Our IHC results showed that the BMP4 protein was expressed in the epidermis and root sheath of the Kazakh sheep skin. The Noggin protein was expressed in the epidermis, root sheath, hair shaft, and dermal papilla of the Kazakh sheep skin. These results provide a theoretical basis for additional studies regarding the association and mechanism of BMP4 and Noggin in coat-color formation in Kazakh sheep. These results may provide new methods for developing treatment strategies for pigmentation disorders and diseases.


CircPSM3 inhibits the proliferation and differentiation of OA chondrocytes by targeting miRNA-296-5p.

Osteoarthritis (OA) is a common chronic bone and joint disease. Circular RNA is a type of non-coding RNA that forms a circular structure with covalent bonds. There is growing evidence that circRNA can function as a functional RNA and play an important role in the occurrence and development of osteoarthritis chondrocytes. However, the exact role of circRNA on OA remains to be studied.Quantificational real-time polymerase chain reaction (qRT-PCR) was used to determine the expression levels of CircPSM3 and miRNA-296-5p in OA chondrocytes. Cell proliferation was detected by the Cell Counting Kit (CCK8), and BMP2, BMP4, BMP6 and RUN2 molecular levels in OA chondrocytes were detected by qRT-PCR and Western Blot (WB). Direct targets of CircPSM3 and miRNA-296-5p in OA chondrocytes were measured by Luciferase reporter assay.CircPSM3 expression was upregulated in OA cartilage tissue and cells. Low expression of CircPSM3 promoted the proliferation and cell differentiation of OA chondrocytes. Meanwhile, miRNA-296-5p was down-regulated in OA cartilage tissue and cells. The Luciferase reporter gene showed that CircPSM3 could target miRNA-296-5p.

The expression level of CircPSM3 and miRNA-296-5p showed a negative correlation. Further research found that a high expression of miRNA-296-5p could effectively promote the proliferation and cell differentiation of OA chondrocytes. Furthermore, miRNA-296-5p inhibitors reversed the effect of si-CircPSM3 on the proliferation and differentiation of OA chondrocytes, while miRNA-296-5p inhibitors enhanced the effect of si-CircPSM3 on the proliferation and differentiation of OA chondrocytes.CircPSM3 was upregulated in OA chondrocytes. CircPSM3 participated in the proliferation and differentiation of OA chondrocytes through targeted binding to miRNA-296-5p. CircPSM3 may become a potential therapeutic target for osteoarthritis treatment.


Induction and maintenance of specific multipotent progenitor stem cells synergistically mediated by Activin A and BMP4 signaling.

We recently reported that epiblast stem cells (EpiSCs)-like cells could be derived from preimplantation embryos (named as AFSCs). Here, we established AFSCs from pre-implantation embryos of multiple mouse strains and showed that unlike EpiSCs, the derivation efficiency of AFSCs was affected by the genetic background. We then used AFSCs lines to dissect the roles of Activin A (Act A) and basic fibroblast growth factor and reported that Act A alone was capable of maintaining self-renewal but not developmental potential in vivo. Finally, we established a novel experimental system, in which AFSCs were efficiently converted to multipotent progenitor stem cells using Act A and bone morphogenetic protein 4 (named as ABSCs). Importantly, these ABSCs contributed to neural mesodermal progenitors and lateral plate mesoderm in postimplantation chimeras.

Taken together, our study established a robust experimental system for the generation of specific multipotent progenitor stem cells that was self-renewable and capable of contributing to embryonic and extra-embryonic tissues.


MicroRNA‑431 inhibits the expression of surfactant proteins through the BMP4/activin/TGF‑β signaling pathway by targeting SMAD4.

The synthesis and secretion of surfactant proteins (SPs) is an important sign of lung maturation. Furthermore, the morbidity of lung developmental diseases, including respiratory distress syndrome and bronchopulmonary dysplasia which are mainly caused by immature lung development and lack of SPs, is increasing. As is well known, multiple microRNAs (miRs/miRNAs) are able to influence lung development via numerous different signaling pathways. However, few studies examine the association between the miRNAs and lung developmental diseases. A previous study has demonstrated that miR‑431 was significantly (F=33.49; P<0.001) downregulated in the lung tissues of Sprague‑Dawley rats at 3 time points, embryonic day 19, embryonic day 21 and postnatal day 3.

The present study reported that the regulation of miR‑431 may influence the expression of SPs. Thus, the further potential mechanisms of miR‑431 in negatively regulating lung development were examined in the present study. Stable A549 cell lines overexpressing or knocking down SMAD family member 4 (SMAD4) transfected with miR‑431 overexpressed or knocked down, and their control groups were established. Subsequently, the expression of bone morphogenetic protein 4 (BMP4), SMAD4 and SPs (SP‑A, SP‑B and SP‑C) at the RNA and protein levels were validated respectively by reverse transcription quantitative PCR and western blotting. miR‑431 exhibited a decreased expression, while BMP4 and SPs exhibited increased expression at the mRNA and protein levels in the SMAD4 knockdown group. Meanwhile, the expression of SPs were reduced in the SMAD4‑knockdown group via overexpressing miR‑431 and increased in the SMAD4‑overexpression group via inhibiting miR‑431. The present results indicate that SMAD4 negatively regulates the expression of SPs, and that miR‑431 negatively regulates the expression of SPs through inhibiting the BMP4/activin/transforming growth factor‑β signaling pathway by targeting SMAD4.


Impact of long-lasting spontaneous physical activity on bone morphogenetic protein 4 in the heart and tibia in murine model of heart failure.

Bone morphogenetic protein 4 (BMP4) plays an important role in bone remodeling and in heart failure pathogenesis. The aim of this study was to evaluate the effect of spontaneous physical activity on the expression of BMP4 in the heart and tibia of the transgenic (Tgαq44) mice, representing a model of chronic heart failure. Tgαq44 and wild-type FVB mice (WT) were randomly assigned either to sedentary or to trained groups undergoing 8 weeks of spontaneous wheel running.

The BMP4 protein expression in heart and tibiae was evaluated using Western immunoblotting and the phosphorus and calcium in the tibiae was assessed using the X-ray microanalysis. BMP4 content in the hearts of the Tgαq44-sedentary mice was by ~490% higher than in the WT-sedentary mice, whereas in tibiae the BMP4 content of the Tgαq44-sedentary mice was similar to that in the WT-sedentary animals. Tgαq44 mice revealed by ~28% poorer spontaneous physical activity than the WT mice. No effect of performed physical activity on the BMP4 content in the hearts of either in the Tgαq44 or WT mice was observed. However, 8-week spontaneous wheel running resulted in a decrease in the BMP4 expression in tibiae (by ~43%) in the group of Tgαq*44 mice only, with no changes in their bone phosphorus and calcium contents. We have concluded that prolonged period of spontaneous physical exercise does not increase the risk of the progression of the BMP4-mediated pathological cardiac hypertrophy and does not affect bone mineral status in the chronic heart failure mice.


FTO Intronic SNP Strongly Influences Human Neck Adipocyte Browning Determined by Tissue and PPARγ Specific Regulation: A Transcriptome Analysis.

Brown adipocytes, abundant in deep-neck (DN) area in humans, are thermogenic with anti-obesity potential. FTO pro-obesity rs1421085 T-to-C single-nucleotide polymorphism (SNP) shifts differentiation program towards white adipocytes in subcutaneous fat. Human adipose-derived stromal cells were obtained from subcutaneous neck (SC) and DN fat of nine donors, of which 3-3 carried risk-free (T/T), heterozygous or obesity-risk (C/C) FTO genotypes. They were differentiated to white and brown (long-term Peroxisome proliferator-activated receptor gamma (PPARγ) stimulation) adipocytes; then, global RNA sequencing was performed and differentially expressed genes (DEGs) were compared. DN and SC progenitors had similar adipocyte differentiation potential but differed in DEGs.

DN adipocytes displayed higher browning features according to ProFAT or BATLAS scores and characteristic DEG patterns revealing associated pathways which were highly expressed (thermogenesis, interferon, cytokine, and retinoic acid, with UCP1 and BMP4 as prominent network stabilizers) or downregulated (particularly extracellular matrix remodeling) compared to SC ones. Part of DEGs in either DN or SC browning was PPARγ-dependent. Presence of the FTO obesity-risk allele suppressed the expression of mitochondrial and thermogenesis genes with a striking resemblance between affected pathways and those appearing in ProFAT and BATLAS, underlining the importance of metabolic and mitochondrial pathways in thermogenesis. Among overlapping regulatory influences that determine browning and thermogenic potential of neck adipocytes, FTO genetic background has a thus far not recognized prominence.


Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction.

Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4.

Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.


Embryology and Morphological (Mal)Development of UPJ.

Kidney parenchyma and collecting system arise from two different embryologic units as a result of a close interaction between them. Therefore, their congenital abnormalities are classified together under the same heading named CAKUT (congenital abnormalities of the kidney and urinary tract). The pathogenesis of CAKUT is thought to be multifactorial. Ureteropelvic junction obstruction (UPJO) is the most common and most investigated form of CAKUT. Despite years of experimental and clinical research, and the information gained on the embryogenesis of the kidney; its etiopathogenesis is still unclear. It involves both genetic and environmental factors. Failure in development of the renal pelvis, failure in the recanalization of ureteropelvic junction, abnormal pyeloureteral innervation, and impaired smooth muscle differentiation are the main proposed mechanisms for the occurrence of UPJO.

There are also single gene mutations like AGTR2, BMP4, Id2 proposed in the etiopathogenesis of UPJO.


Effect for Human Genomic Variation During the BMP4-Induced Conversion From Pluripotent Stem Cells to Trophoblast.

The role of genomic variation in differentiation is currently not well understood. Here, the genomic variations were determined with the whole-genome sequencing for three pairs of pluripotent stem cell lines and their corresponding BMP4-induced trophoblast cell lines. We identified ∼3,500 single nucleotide variations and ∼4,500 indels by comparing the genome sequenced data between the stem cell lines and the matched BMP4-induced trophoblast cell lines and annotated them by integrating the epigenomic and transcriptomic datasets. Relatively, introns enrich more variations. We found ∼45% (42 genes) of the differentially expressed genes in trophoblasts that associate genomic variations. Six variations, located at transcription factor binding sites where H3K4me3 and H3K27ac are enriched in both H1 and H1_BMP4, were identified. The epigenetic status around the genomic variations in H1 was similar to that in H1_BMP4. This means that the variation-associated gene’s expression change can not be attributed to epigenetic alteration. The genes associated with the six variations were upregulated in differentiation. We inferred that during the differentiation, an increased in the expression level of the MEF2C gene is due to a genomic variation in chromosomes 5: 88179358 A > G, which is at a binding site of TFs KLF16, NR2C2, and ZNF740 to MEF2C. Allele G shows a higher affinity to the TFs in the induced cells. The increased expression of MEF2C leads to an increased expression of TF MEF2C’s target genes, subsequently affecting the differentiation.

Although genomic variation should not be a dominant factor in differentiation, we believe that genomic variation could indeed play a role in the differentiation from stem cells into trophoblast.

Lieven Gevaert, Bio-engineer


In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like.

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

Neural tube defects are the most common severe central nervous system anomalies only second to cardiovascular abnormalities resulting in congenital morbidity and mortality. The nervous system is ectodermal in origin. The central nervous system consists of the brain and spinal cord formed by folding of dorsal part neural plates under the influence of underlying notochord and prechordal mesoderm and closure anterior (cranial) and posterior (caudal) neuropores by a process called neurulation that begins as early as 3 and 4 weeks of conception. Failure to complete of neurulation results in neural tube defects (NTDs). Neurulation consists of two phases i.e., primary and secondary neurulation. Primary neurulation is defined by folding of the dorsal part of the neural tube and lengthening of neural plates in the longitudinal axis and narrowing of the cross-section by the phenomenon called convergent extension forming the brain and spinal cord. Primary neurulation is followed by canalization of neural tubes, forming a distal part of the spinal cord called secondary neurulation.[1] Fibroblast growth factor (FGF) signaling concordant with suppression of bone morphogenetic protein 4 (BMP4), a transforming growth factor induces the formation of the neural plate. Also, retinoic acid organizes the cranial-caudal axis by regulating the expression of homeobox genes.[2] Neural tube defects can be present anywhere from the brain to the end of the spinal cord. Open NTDs are due to failure of primary neuralation and associated with hydrocephalus, Chiari II malformation, etc. Neural tissue is exposed to and associated with cerebrospinal fluid (CSF) leakage. Closed NTDs are due to failure of secondary neuralation and are generally confined to the Spinal cord. Neural tissue is not exposed. The closed neural tube defects occur post neurulation and include lipoma with a dorsal defect (lipomyelomeningocele, lipomyelocele), especially when a subcutaneous mass is present. Common variants of NTDs are as follows: 1. Spina bifida occulta: failure of caudal neuropore to close. The spinal cord, meninges, and overlying skin remain intact, with no herniation. 2. Spina bifida cystica: meningocele (herniation of meninges only) and myelomeningocele (herniation of both meninges and neural tissue) 3. Myeloschisis: exposed neural tissue without skin or meninges covering. 4. Anencephaly: failure of rostral neuropore to close thus the brain and cranial vault are grossly malformed with normal hindbrain development.


High SET Domain Bifurcated 1 (SETDB1) Expression Predicts Poor Prognosis in Breast Carcinoma.

BACKGROUND SETDB1, an H3K9-specific histone methyltransferase, plays important roles in the progression of various human cancers. However, the expression patterns and its clinical roles of SETDB1 remain elusive in breast cancer (BC). MATERIAL AND METHODS The transcriptional level of SETDB1 and survival data in BC were analyzed through UALCAN, ONCOMINE, and Pan Cancer Prognostics Database. SETDB1 protein expression was assessed by immunohistochemistry (IHC) in 159 BC tissue samples. The associations between SETDB1 expression and clinical pathological characteristics of patients were analyzed. The GEO dataset GSE108656 was downloaded and analyzed to identify the differentially expressed genes (DEGs) between control and BC cells targeting interference with SETDB. The DEGs were further integrated by bioinformatics analysis to decipher the key signaling pathways and hub genes that are regulated by SETDB. RESULTS The public databases showed the level of SETDB1 mRNA was significantly upregulated in BC. Our IHC results demonstrated the level of SETDB1 protein was associated with tumor size (P=0.028), histopathological grading (P=0.012), lymph node metastasis (P<0.001), and TNM stage (P<0.001). High expression of SETDB1 indicated worse overall survival (P=0.015) and shorter relapse-free survival (P=0.027). The bioinformatic analysis of GSE108656 suggested that the SETDB1-related DEGs was mainly enriched in antigen processing and presentation, as well as immune networks in BC. The cytoHubba analysis suggested the top 10 hub genes were IL6, BMP4, CD74, PECAM1, HLA-DPA1, HLA-DRA, LAMC1, CTSB, SERPINA1, and CTSD. CONCLUSIONS The results suggest that SETDB1 is an oncogene and can serve as a prognostic biomarker for BC. However, the mechanisms of SETDB1 in BC remain to be explored.


A Boolean Logical model for Reprogramming of Testes-derived male Germline Stem Cells into Germline pluripotent stem cells.

Male germline stem (GS) cells are responsible for the maintenance of spermatogenesis throughout the adult life of males. Upon appropriate in vitro culture conditions, these GS cells can undergo reprogramming to become germline pluripotent stem (GPS) cells with the loss of spermatogenic potential. In recent years, voluminous data of gene transcripts in GS and GPS cells have become available. However, the mechanism of reprogramming of GS cells into GPS cells remains elusive. This study was designed to develop a Boolean logical model of gene regulatory network (GRN) that might be involved in the reprogramming of GS cells into GPS cells.The gene expression profile of GS and GPS cells (GSE ID: GSE11274 and GSE74151) were analyzed using R Bioconductor to identify differentially expressed genes (DEGs) and were functionally annotated with DAVID server. Potential pluripotent genes among the DEGs were then predicted using a combination of machine learning [Support Vector Machine (SVM)] and BLAST search. Protein isoforms were identified by pattern matching with UniProt database with in-house scripts written in C++. Both linear and non-linear interaction maps were generated using the STRING server. CellNet is used to study the relationship of GRNs between the GS and GPS cells. Finally, the GRNs involving all the genes from integrated methods and literature was constructed and qualitative modelling for reprogramming of GS to GPS cells were done by considering the discrete, asynchronous, multivalued logical formalism using the GINsim modeling and simulation tool.Through the use of machine learning and logical modeling, the present study identified 3585 DEGs and 221 novel pluripotent genes including Tet1, Cdh1, Tfap2c, Etv4, Etv5, Prdm14, and Prdm10 in GPS cells. Pathway analysis revealed that important signaling pathways such as core pluripotency network, PI3K-Akt, WNT, GDNF and BMP4 signalling pathways were important for the reprogramming of GS cells to GPS cells. On the other hand, CellNet analysis of GRNs of GS and GPS cells revealed that GS cells were similar to gonads whereas GPS cells were similar to ESCs in gene expression profile. A logical regulatory model was developed, which showed that TGFβ negatively regulated the reprogramming of the GS to GPS cells, as confirmed by perturbations studies.The study identified novel pluripotent genes involved in the reprogramming of GS cells into GPS cells. A multivalued logical model of cellular reprogramming is proposed, which suggests that reprogramming of GS cells to GPS cells involves signalling pathways namely LIF, GDNF, BMP4, and TGFβ along with some novel pluripotency genes.


A small nuclear acidic protein (MTI-II, Zn2+-binding protein, parathymosin) attenuates TNF-α inhibition of BMP-induced osteogenesis by enhancing accessibility of the Smad4-NF-κB p65 complex to Smad binding element.

Pro-inflammatory cytokines prevent bone regeneration in vivo and activation of nuclear factor-κB (NF-κB) signaling has been proposed to lead to suppression of bone morphogenetic protein (BMP)-induced osteogenesis via direct binding of p65 to Smad4 in vitro. Application of a small nuclear acidic protein (MTI-II) and its delivered peptide, MPAID (MTI-II peptide anti-inflammatory drug) has been described to elicit therapeutic potential via strong anti-inflammatory action following the physical association of MTI-II and MPAID with p65. However, it is unclear whether MTI-II attenuates tumor necrosis factor (TNF)-α inhibition of BMP-induced osteogenesis. Herein, we found that TNF-α-mediated suppression of responses associated with BMP4-induced osteogenesis, including expression of the osteocalcin encoding gene Ocn, Smad binding element (SBE)-dependent luciferase activity, alkaline phosphatase activity, and alizarin red S staining were largely restored by MTI-II and MPAID in MC3T3-E1 cells. Mechanistically, MTI-II and MPAID did not inhibit nuclear translocation of p65 or disassociate Smad4 from p65. Further, results from chromatin immunoprecipitation (ChIP) analyses revealed that Smad4 enrichment in cells over-expressing MTI-II and treated with TNF-α was equivalent to that in cells without TNF-α treatment. Alternatively, Smad4 enrichment was considerably decreased following TNF-α treatment in control cells. Moreover, p65 enrichment in the Id-1 promoter SBE was detected only when cells over-expressing MTI-II were stimulated with TNF-α. Overall, our study concludes that MTI-II restored TNF-α-inhibited suppression of BMP-Smad-induced osteogenic differentiation by enhancing accessibility of the Smad4-p65 complex to the SBE rather than by liberating Smad4 from p65.


GATA3 Mediates a Fast, Irreversible Commitment to BMP4-Driven Differentiation in Human Embryonic Stem Cells.

During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling.