Divergent and Compensatory Effects of BMP2 and BMP4 on the VSMC Phenotype and BMP4's Role in Thoracic Aortic Aneurysm Development.

Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.

Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. In situ hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

Epithelial-mesenchymal interaction protects normal colonocytes from 4-HNE-induced phenotypic transformation.

INTRODUCTION: Recent studies have shown that epithelial-stromal interactions could play a role in the development of colorectal cancer. Here, we investigated the role of fibroblasts in the transformation of normal colonocytes induced by 4-HNE. METHODS: Normal Co colonocytes and nF fibroblasts from the same mouse colon were exposed, in monoculture (m) or coculture (c), to 4-HNE (5 µM) twice weekly for 3 weeks. Gene expression was then analysed and the ability of Co colonocytes to grow in anchorage-independent conditions was tested in soft agar. Fibroblasts previously treated or not with 4-HNE were also seeded in culture inserts positioned above the agar layers to allow paracrine exchanges with colonocytes. RESULTS: First, 60% of the genes studied were modulated by coculture in Co colonocytes, with notably increased expression of BMP receptors. Furthermore, while 4-HNE increased the ability of monoculture-treated Co colonocytes to form colonies, this effect was not observed in coculture-treated Co colonocytes. Adding a selective BMPR1 inhibitor during the treatment phase abolished the protective effect of coculture. Conversely, addition of a BMP4 agonist to the medium of monoculture-treated Co colonocytes prevented phenotypic transformation by 4-HNE. Second, the presence of nF(m)-HNE fibroblasts during the soft agar assay increased the number and size of Co(m) colonocyte colonies, regardless of whether these cells had been previously treated with 4-HNE in monoculture. For soft agar assays performed with nF(c) and Co(c) cells initially treated in coculture, only the reassociation between Co(c)-HNE and nF(c)-HNE resulted in a small increase in the number of colonies. CONCLUSIONS: During the exposure phase, the epithelial-mesenchymal interaction protected colonocytes from 4-HNE-induced phenotypic transformation via activation of the BMP pathway. This intercellular dialogue also limited the ability of fibroblasts to subsequently promote colonocyte-anchorage-independent growth. In contrast, fibroblasts pre-exposed to 4-HNE in monoculture strongly increased the ability of Co(m) colonocytes to form colonies.

Small Molecules Promote the Rapid Generation of Dental Epithelial Cells from Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.

Osteogenic Effects of the Diospyros lotus L. Leaf Extract on MC3T3-E1 Pre-Osteoblasts and Ovariectomized Mice via BMP2/4 and TGF ß Pathways.

Osteoporosis, a disease defined by the primary bone strength due to a low bone mineral density, is a bone disorder associated with increased mortality in the older adult population. Osteoporosis is mainly treated via hormone replacement therapy, bisphosphates, and anti-bone resorption agents. However, these agents exert severe side effects, necessitating the development of novel therapeutic agents. Many studies are focusing on osteogenic agents as they increase the bone density, which is essential for osteoporosis treatment. Here, we aimed to investigate the effects of Diospyros lotus L. leaf extract (DLE) and its components on osteoporosis in MC3T3-E1 pre-osteoblasts and ovariectomized mice and to elucidate the underlying related pathways. DLE enhanced the differentiation of MC3T3-E1 pre-osteoblasts, with a 1.5-fold elevation in ALP activity, and increased the levels of osteogenic molecules, RUNX family transcription factor 2, and osterix. This alteration resulted from the activation of bone morphogenic protein 2/4 (BMP2/4) and transformation of growth factor ß (TGF ß) pathways. In ovariectomized mice, DLE suppressed the decrease in bone mineral density by 50% and improved the expression of other bone markers, which was confirmed by the 3~40-fold increase in osteogenic proteins and mRNA expression levels in bone marrow cells. The three major compounds identified in DLE exhibited osteogenic and estrogenic activities with their aglycones, as previously reported. Among the major compounds, myricitrin alone was not as strong as whole DLE with all its constituents. The osteogenic activity of DLE was partially suppressed by the inhibitor of estrogen signaling, indicating that the estrogenic activity of DLE participated in its osteogenic activity. Overall, DLE suppresses osteoporosis by inducing osteoblast differentiation.

BMP4 in Human Endometrial Stromal Cells Can Affect Decidualization by Regulating FOXO1 Expression.

CONTEXT: Recurrent spontaneous abortion (RSA) is defined as the loss of 2 or more consecutive intrauterine pregnancies with the same sexual partner in the first trimester. Despite its significance, the etiology and underlying mechanisms of RSA remain elusive. Defective decidualization is proposed as one of the potential causes of RSA, with abnormal decidualization leading to disturbances in trophoblast invasion function. OBJECTIVE: To assess the role of bone morphogenetic protein 4 (BMP4) in decidualization and RSA. METHODS: Decidual samples were collected from both RSA patients and healthy controls to assess BMP4 expression. In vitro cell experiments utilized the hESC cell line to investigate the impact of BMP4 on decidualization and associated aging, as well as its role in the maternal-fetal interface communication. Subsequently, a spontaneous abortion mouse model was established to evaluate embryo resorption rates and BMP4 expression levels. RESULTS: Our study identified a significant downregulation of BMP4 expression in the decidua of RSA patients compared to the normal control group. In vitro, BMP4 knockdown resulted in inadequate decidualization and inhibited associated aging processes. Mechanistically, BMP4 was implicated in the regulation of FOXO1 expression, thereby influencing decidualization and aging. Furthermore, loss of BMP4 hindered trophoblast migration and invasion via FOXO1 modulation. Additionally, BMP4 downregulation was observed in RSA mice. CONCLUSION: Our findings highlighted the downregulation of BMP4 in both RSA patients and mice. BMP4 in human endometrial stromal cells was shown to modulate decidualization by regulating FOXO1 expression. Loss of BMP4 may contribute to the pathogenesis of RSA, suggesting potential avenues for abortion prevention strategies.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Pilose antler extract promotes hair growth in androgenic alopecia mice by promoting the initial anagen phase.

Androgenetic alopecia (AGA) is a prevalent disease in worldwide, local application or oral are often used to treat AGA, however, effective treatments for AGA are currently limited. In this work, we observed the promoting the initial anagen phase effect of pilose antler extract (PAE) on hair regeneration in AGA mice. We found that PAE accelerated hair growth and increased the degree of skin blackness by non-invasive in vivo methods including camera, optical coherence tomography and dermoscopy. Meanwhile, HE staining of sagittal and coronal skin sections revealed that PAE augmented the quantity and length of hair follicles, while also enhancing skin thickness and hair papilla diameter. Furthermore, PAE facilitated the shift of the growth cycle from the telogen to the anagen phase and expedited the proliferation of hair follicle stem cells and matrix cells in mice with AGA. This acceleration enabled the hair follicles to enter the growth phase at an earlier stage. PAE upregulated the expression of the sonic hedgehog (SHH), smoothened receptor, glioma-associated hemolog1 (GLI1), and downregulated the expression of bone morphogenetic protein 4 (BMP4), recombinant mothers against decapentaplegic homolog (Smad) 1 and 5 phosphorylation. This evidence suggests that PAE fosters hair growth and facilitates the transition of the growth cycle from the telogen to the anagen phase in AGA mice. This effect is achieved by enhancing the proliferation of follicle stem cells and matrix cells through the activation of the SHH/GLI pathway and suppression of the BMP/Smad pathway.

SHH regulates penile morphology and smooth muscle through a mechanism involving BMP4 and GREM1.

BACKGROUND: The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with erectile dysfunction (ED), initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle and increased collagen, which alter penile architecture and make corpora cavernosa smooth muscle less able to relax in response to neurotransmitters, resulting in ED. AIM: Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses penile remodeling after CN injury through an unknown mechanism; we examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1). METHODS: Primary cultures of smooth muscle cells were established from prostatectomy, diabetic, hypertension and Peyronie's (control) (N = 18) patients. Cultures were characterized by ACTA2, CD31, P4HB, and nNOS immunohistochemical analysis. Patient smooth muscle cell growth was quantified in response to BMP4 and GREM1 treatment. Adult Sprague Dawley rats underwent 1 of 3 surgeries: (1) uninjured or CN-injured rats were treated with BMP4, GREM1, or mouse serum albumin (control) proteins via Affi-Gel beads (N = 16) or peptide amphiphile (PA) (N = 26) for 3 and 14 days, and trichrome stain was performed; (2) rats underwent sham (N = 3), CN injury (N = 9), or CN injury and SHH PA treatment for 1, 2, and 4 days (N = 9). OUTCOMES: Western analysis for BMP4 and GREM1 was performed; (3) rats were treated with 5E1 SHH inhibitor (N = 6) or IgG (control; N = 6) for 2 and 4 days, and BMP4 and GREM1 localization was examined. Statistics were performed by analysis of variance with Scheffé's post hoc test. RESULTS: BMP4 increased patient smooth muscle cell growth, and GREM1 decreased growth. In rats, BMP4 treatment via Affi-Gel beads and PA increased smooth muscle at 3 and 14 days of treatment. GREM1 treatment caused increased collagen and smooth muscle at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1, while SHH PA altered Western band size, suggesting alternative cleavage and range of BMP4 and GREM1 signaling. SHH inhibition in rats increased BMP4 and GREM1 in fibroblasts. CLINICAL IMPLICATIONS: Understanding how SHH PA preserves and regenerates penile morphology after CN injury will aid development of ED therapies. STRENGTHS AND LIMITATIONS: SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. CONCLUSION: Part of the mechanism of how SHH regulates corpora cavernosa smooth muscle involves BMP4 and GREM1.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

An Hsp70 promoter-based mouse for heat shock-induced gene modulation.

Physical therapy is extensively employed in clinical settings. Nevertheless, the absence of suitable animal models has resulted in an incomplete understanding of the in vivo mechanisms and cellular distribution that respond to physical stimuli. The objective of this research was to create a mouse model capable of indicating the cells affected by physical stimuli. In this study, we successfully established a mouse line based on the heat shock protein 70 (Hsp70) promoter, wherein the expression of CreERT2 can be induced by physical stimuli. Following stimulation of the mouse tail, ear, or cultured calvarias with heat shock (generated by heating, ultrasound, or laser), a distinct Cre-mediated excision was observed in cells stimulated by these physical factors with minimal occurrence of leaky reporter expression. The application of heat shock to Hsp70-CreERT2; FGFR2-P253R double transgenic mice or Hsp70-CreERT2 mice infected with AAV-BMP4 at calvarias induced the activation of Cre-dependent mutant FGFR2-P253R or BMP4 respectively, thereby facilitating the premature closure of cranial sutures or the repair of calvarial defects. This novel mouse line holds significant potential for investigating the underlying mechanisms of physical therapy, tissue repair and regeneration, lineage tracing, and targeted modulation of gene expression of cells in local tissue stimulated by physical factor at the interested time points. KEY MESSAGES: In the study, an Hsp70-CreERT2 transgenic mouse was generated for heat shock-induced gene modulation. Heat shock, ultrasound, and laser stimulation effectively activated Cre expression in Hsp70-CreERT2; reporter mice, which leads to deletion of floxed DNA sequence in the tail, ear, and cultured calvaria tissues of mice. Local laser stimuli on cultured calvarias effectively induce Fgfr2-P253R expression in Hsp70-mTmG-Fgfr2-P253R mice and result in accelerated premature closure of cranial suture. Heat shock activated AAV9-FLEX-BMP4 expression and subsequently promoted the repair of calvarial defect of Hsp70-CreERT2; Rosa26-mTmG mice.

Cdx1 and Gsc distinctly regulate the transcription of BMP4 target gene ventx3.2 by directly binding to the proximal promoter region in Xenopus gastrulae.

A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide chromatin immunoprecipitation-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, the chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral marginal zone and dorsal marginal zone, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the ventral marginal zone and dorsal marginal zone by directly binding to the promoter region during Xenopus gastrulation.

Directed osteogenic differentiation of human bone marrow mesenchymal stem cells via sustained release of BMP4 from PBVHx-based nanoparticles.

Bone Morphogenetic Protein 4 (BMP4) is crucial for bone and cartilage tissue regeneration, essential in medical tissue engineering, cosmetology, and aerospace. However, its cost and degradation susceptibility pose significant clinical challenges. To enhance its osteogenic activity while reducing dosage and administration frequency, we developed a novel long-acting BMP4 delivery system using poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PBVHx) nanoparticles with soybean lecithin-modified BMP4 (sBP-NPs). These nanoparticles promote directed osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through sustained BMP4 release. sBP-NPs exhibited uniform size (100-200 nm) and surface charges, with higher BMP4 entrapment efficiency (82.63 %) compared to controls. After an initial burst release within 24 h, sBP-NPs achieved 80 % cumulative BMP4 release within 20 days, maintaining levels better than control BP-NPs with unmodified BMP4. Co-incubation and nanoparticle uptake experiments confirmed excellent biocompatibility of sBP-NPs, promoting hBMSC differentiation towards osteogenic lineage with increased expression of type I collagen, calcium deposition, and ALP activity (> 20,000 U/g protein) compared to controls. Moreover, hBMSCs treated with sBP-NPs exhibited heightened expression of osteogenic genetic markers, surpassing control groups. Hence, this innovative strategy of sustained BMP4 release from sBP-NPs holds potential to revolutionize bone regeneration in minimally invasive surgery, medical cosmetology or space environments.

AEBP1 promotes papillary thyroid cancer progression by activating BMP4 signaling.

Papillary thyroid cancer (PTC) is the most prevalent endocrine cancer worldwide. Approximately 30 % of PTC patients will progress into the advanced or metastatic stage and have a relatively poor prognosis. It is well known that epithelial-mesenchymal transition (EMT) plays a pivotal role in thyroid cancer metastasis, resistance to therapy, and recurrence. Clarifying the molecular mechanisms of EMT in PTC progression will help develop the targeted therapy of PTC. The aberrant expression of some transcription factors (TFs) participated in many pathological processes of cancers including EMT. In this study, by performing bioinformatics analysis, adipocyte enhancer-binding protein 1 (AEBP1) was screened as a pivotal TF that promoted EMT and tumor progression in PTC. In vitro experiments indicated that knockout of AEBP1 can inhibit the growth and invasion of PTC cells and reduce the expression of EMT markers including N-cadherin, TWIST1, and ZEB2. In the xenograft model, knockout of AEBP1 inhibited the growth and lung metastasis of PTC cells. By performing RNA-sequencing, dual-luciferase reporter assay, and chromatin immunoprecipitation assay, Bone morphogenetic protein 4 (BMP4) was identified as a downstream target of AEBP1. Over-expression of BMP4 can rescue the inhibitory effects of AEBP1 knockout on the growth, invasion, and EMT phenotype of PTC cells. In conclusion, these findings demonstrated that AEBP1 plays a critical role in PTC progression by regulating BMP4 expression and the AEBP1-BMP4 axis may present novel therapeutic targets for PTC treatment.

Ginsenosides in Panax ginseng Extract Promote Anagen Transition by Suppressing BMP4 Expression and Promote Human Hair Growth by Stimulating Follicle-Cell Proliferation.

Studies showing that Panax ginseng promotes hair growth have largely been conducted using mice; there are few reports on how P. ginseng affects human hair growth. In particular, little is known about its effect on the telogen to anagen transition. To determine the effect of P. ginseng on human hair growth and the transition from the telogen to the anagen phase. The effects of P. ginseng extract (PGE) and the three major ginsenoside components, Rb1, Rg1, and Re, on the proliferation of human dermal papilla cells (DPCs) and human outer root sheath cells (ORSCs) were investigated. The effects of these compounds on the cell expression of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 18 (FGF18) and Noggin were assessed by real-time PCR. The effect of PGE on hair-shaft elongation was determined in a human hair follicle organ-culture system. PGE and the three ginsenosides stimulated the proliferation of DPCs and ORSCs and suppressed BMP4 expression in DPCs but did not affect FGF18 expression in ORSCs and Noggin expression in DPCs. PGE stimulated hair-shaft growth. PGE and the ginsenosides Rb1, Rg1, and Re stimulate the transition from the telogen phase to anagen phase of the hair cycle by suppressing BMP4 expression in DPCs. These compounds might be useful for promoting the growth of human hair.

Methotrexate inhibits BMP4 and abrogates the hypertrophic chondrocyte phenotype of synovial fibroblasts in juvenile idiopathic arthritis.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS. METHODS: We selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4. RESULTS: ColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells. CONCLUSIONS: Methotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation.

Comparison of Four Protocols for In Vitro Differentiation of Human Embryonic Stem Cells into Trophoblast Lineages by BMP4 and Dual Inhibition of Activin/Nodal and FGF2 Signaling.

Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments.

Typical neonicotinoids and organophosphate esters, but not their metabolites, adversely impact early human development by activating BMP4 signaling.

Recent studies have highlighted the presence of potentially harmful chemicals, such as neonicotinoids (NEOs) and organophosphate esters (OPEs), in everyday items. Despite their potential threats to human health, these dangers are often overlooked. In a previous study, we discovered that NEOs and OPEs can negatively impact development, but liver metabolism can help mitigate their harmful effects. In our current research, our objective was to investigate the toxicity mechanisms associated with NEOs, OPEs, and their liver metabolites using a human embryonic stem cell-based differentiation model that mimics early embryonic development. Our transcriptomics data revealed that NEOs and OPEs significantly influenced the expression of hundreds of genes, disrupted around 100 biological processes, and affected two signaling pathways. Notably, the BMP4 signaling pathway emerged as a key player in the disruption caused by exposure to these pollutants. Both NEOs and OPEs activated BMP4 signaling, potentially impacting early embryonic development. Interestingly, we observed that treatment with a human liver S9 fraction, which mimics liver metabolism, effectively reduced the toxic effects of these pollutants. Most importantly, it reversed the adverse effects dependent on the BMP4 pathway. These findings suggest that normal liver function plays a crucial role in detoxifying environmental pollutants and provides valuable experimental insights for addressing this issue.

BMP4 up-regulated by 630 nm LED irradiation is associated with the amelioration of rheumatoid arthritis.

Rheumatoid arthritis (RA) is caused by inflammatory response of joints with cartilage and damage of synovium and bone erosion. In our previous studies, it has showed that irradiation of 630 nm LED reduce inflammation of synovial fibroblasts and cartilage and bone destruction in RA. However, the key genes and mechanism in ameliorating RA by irradiation of 630 nm LED remains unknown. In this study, human fibroblast-like synoviocytes (FLS) cell line MH7A and primary human RA-FLSs were treated with TNF-α and 630 nm LED irradiation with the different energy density. The mRNA sequencing was performed to screen the differentially expressed genes (DEGs). In all datasets, 10 DEGs were identified through screening. The protein interaction network analysis showed that 8 out of the 10 DEGs interacted with each other including IL-6, CXCL2, CXCL3, MAF, PGF, IL-1RL1, RRAD and BMP4. This study focused on BMP4, which is identified as important morphogens in regulating the development and homeostasis. CCK-8 assay results showed that 630 nm LED irradiation did not affect the cell viability. The qPCR and ELISA results showed that TNF-α stimulation inhibited BMP4 mRNA and protein level and irradiation of 630 nm LED increased the BMP4 mRNA and protein level in MH7A cells. In CIA and transgenic hTNF-α mice models, H&E staining showed that irradiation of 630 nm LED decreased the histological scores assessed from inflammation and bone erosion, while BMP4 expression level was up-regulated after 630 nm LED irradiation. Pearson correlation analysis shown that BMP4 protein expression was negatively correlated with the histological score of CIA mice and transgenic hTNF-α mice. These results indicated that BMP4 increased by irradiation of 630 nm LED was associated with the amelioration of RA, which suggested that BMP4 may be a potential targeting gene for photobiomodulation.