Bone morphogenetic protein 4 ameliorates bleomycin-induced pulmonary fibrosis in mice by repressing NLRP3 inflammasome activation and epithelial-mesenchymal transition.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and deadly lung disease with limited therapeutic options. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor that belongs to the transforming growth factor-ß superfamily, is able to relieve pulmonary fibrosis in mice; nevertheless, the potential mechanism of action remains largely unknown. Growing evidence supports the notion that reiterant damage to the alveolar epithelial cells (AECs) is usually the "prime mover" for pulmonary fibrosis. Here, we examined the effect and mechanisms of BMP4 on bleomycin (BLM)-induced activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and epithelial-mesenchymal transition (EMT) in vivo and in vitro. Methods: The in vivo impact of BMP4 was investigated in a BLM mouse model. Histopathologic changes were analyzed by hematoxylin-eosin (H&E) and Masson's trichrome staining. The NLRP3 inflammasome activation was determined by quantitative real time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. Biomarkers of EMT were measured by qRT-PCR, Western blot and immunofluorescence staining. The in vitro impact of BMP4 on BLM-induced NLRP3 inflammasome activation and EMT was explored in A549 AECs. We also evaluated whether BMP4 inhibited BLM-activated ERK1/2 signaling to address the possible molecular mechanisms. Results: BMP4 was significantly downregulated in the mouse lungs from BLM-induced pulmonary fibrosis. BMP4+/- mice presented with more severe lung fibrosis in response to BLM, and accelerated NLRP3 inflammasome activation and EMT process compared with that in BMP4+/+ mice. Whereas overexpression of BMP4 by injecting adeno-associated virus (AAV) 9 into mice attenuated BLM-induced fibrotic changes, NLRP3 inflammasome activation, and EMT in the mouse lungs, thus exerting protective efficacy against lung fibrosis. In vitro, BMP4 significantly reduced BLM-induced activation of NLRP3 inflammasome and EMT in human alveolar epithelial A549 cells. Mechanically, BMP4 repressed BLM-induced activation of ERK1/2 signaling in vivo and in vitro, suggesting that ERK1/2 inactivation contributes to BMP4-induced effects on BLM-induced activation of NLRP3 inflammasome and EMT. Conclusions: Our findings suggest that BMP4 can suppress NLRP3 inflammasome activation and EMT in AECs via inhibition of ERK1/2 signaling pathway, thus has a potential for the treatment of pulmonary fibrosis.

Bone morphogenetic protein 4 derived from the cerebrospinal fluid in patients with postherpetic neuralgia induces allodynia via the crosstalk between microglia and astrocyte.

INTRODUCTION: During postherpetic neuralgia (PHN), the cerebral spinal fluid (CSF) possesses the capability to trigger glial activation and inflammation, yet the specific changes in its composition remain unclear. Recent findings from our research indicate elevations of central bone morphogenetic protein 4 (BMP4) during neuropathic pain (NP), serving as an independent modulator of glial cells. Herein, the aim of the present study is to test the CSF-BMP4 expressions and its role in the glial modulation in the process of PHN. METHODS: CSF samples were collected from both PHN patients and non-painful individuals (Control) to assess BMP4 and its antagonist Noggin levels. Besides, intrathecal administration of both CSF types was conducted in normal rats to evaluate the impact on pain behavior, glial activity, and inflammation.; Additionally, both Noggin and STAT3 antagonist-Stattic were employed to treat the PHN-CSF or exogenous BMP4 challenged cultured astrocytes to explore downstream signals. Finally, microglial depletion was performed prior to the PHN-CSF intervention so as to elucidate the microglia-astrocyte crosstalk. RESULTS: BMP4 levels were significantly higher in PHN-CSF compared to Control-CSF (P < 0.001), with a positive correlation with pain duration (P < 0.05, r = 0.502). Comparing with the Control-CSF producing moderate paw withdrawal threshold (PWT) decline and microglial activation, PHN-CSF further exacerbated allodynia and triggered both microglial and astrocytic activation (P < 0.05). Moreover, PHN-CSF rather than Control-CSF evoked microglial proliferation and pro-inflammatory transformation, reinforced iron storage, and activated astrocytes possibly through both SMAD159 and STAT3 signaling, which were all mitigated by the Noggin application (P < 0.05). Next, both Noggin and Stattic effectively attenuated BMP4-induced GFAP and IL-6 upregulation, as well as SMAD159 and STAT3 phosphorylation in the cultured astrocytes (P < 0.05). Finally, microglial depletion diminished PHN-CSF induced astrogliosis, inflammation and endogenous BMP4 expression (P < 0.05). CONCLUSION: Our study highlights the role of CSF-BMP4 elevation in glial activation and allodynia during PHN, suggesting a potential therapeutic avenue for future exploration.

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.

Design and characterization of a novel tumor-homing cell-penetrating peptide for drug delivery in TGFBR3 high-expressing tumors.

Targeted therapy has attracted more and more attention in cancer treatment in recent years. However, due to the diversity of tumor types and the mutation of target sites on the tumor surface, some existing targets are no longer suitable for tumor therapy. In addition, the long-term administration of a single targeted drug can also lead to drug resistance and attenuate drug potency, so it is important to develop new targets for tumor therapy. The expression of Type III transforming growth factor ß receptor (TGFBR3) is upregulated in colon, breast, and prostate cancer cells, and plays an important role in the occurrence and development of these cancers, so TGFBR3 may be developed as a novel target for tumor therapy, but so far there is no report on this research. In this study, the structure of bone morphogenetic protein 4 (BMP4), one of the ligands of TGFBR3 was analyzed through the docking analysis with TGFBR3 and sequence charge characteristic analysis, and a functional tumor-targeting penetrating peptide T3BP was identified. The results of fluorescent labeling experiments showed that T3BP could target and efficiently enter tumor cells with high expression of TGFBR3, especially A549 cells. When the expression of TGFBR3 on the surface of tumor cells (HeLa) was knocked down by RNA interference, the high delivery efficiency of T3BP was correspondingly reduced by 40%, indicating that the delivery was TGFBR3-dependent. Trichosanthin (TCS, a plant-derived ribosome inactivating protein) fused with T3BP can enhance the inhibitory activity of the fusion protein on A549 cells by more than 200 times that of TCS alone. These results indicated that T3BP, as a novel targeting peptide that can efficiently bind TGFBR3 and be used for targeted therapy of tumors with high expression of TGFBR3. This study enriches the supply of tumor-targeting peptides and provides a new potential application option for the treatment of tumors with high expression of TGFBR3.

The serum levels of activin A and bone morphogenetic protein-4 and -6 in patients with fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an ultrarare and disabling genetic disorder of connective tissue characterized by congenital malformation of the great toes, and progressive heterotopic ossification (HO) in soft connective tissues. A gain-of-function mutation of activin A receptor type I (ACVR1) enables ACVR1 to recognize activin A as an agonist with bone morphogenetic protein (BMP) signalling that leads to HO. Previous studies confirmed that activin A stimulates BMP signalling in vitro and drives HO in mouse models of FOP. However, the roles for BMP4 and BMP6 in FOP are supported only by correlative evidence in vitro. Thus, it remains unclear whether the circulating levels of activin A, BMP4 and BMP6 correlate with flare-ups in FOP patients. Hence, we investigated the protein levels of activin A, BMP4 and BMP6 in the serum of FOP patients. RESULTS: We recruited 16 untreated FOP patients and 16 age- and sex- matched healthy control subjects in this study. The 16 FOP patients were retrospectively divided into the flare-up group (n = 8) and remission group (n = 8) depending on whether they had flare-ups or worsening of any joint movement in the last 6 months. The serum activin A, BMP4 and BMP6 levels were detected by enzyme-linked immunosorbent assay. The serum activin A, BMP4 and BMP6 levels were slightly higher in FOP patients (median: 434.05 pg/mL, 459.48 pg/mL and 67.84 pg/mL) versus healthy control subjects (median: 364.14 pg/mL, 450.39 pg/mL and 55.36 pg/mL). However, there were no statistically significant differences between the two groups (p > 0.05 for all items), nor were there significant differences between the flare-up and remission groups of FOP (p > 0.05 for all items). Univariate and multivariate logistic regression analyses showed that age, sex, and serum activin A, BMP4 and BMP6 levels were not related to flare-up in FOP patients. CONCLUSIONS: There were no significant differences in the serum levels of activin A, BMP4 and BMP6 in FOP patients compared with healthy control subjects. Serum activin A, BMP4 and BMP6 proteins might not be the stimulators for FOP flare-up, and may not be biomarkers for FOP diagnosis.

Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells.

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

The role of bone morphogenetic protein 4 in corneal injury repair.

PURPOSE: Corneal injury may cause neovascularization and lymphangiogenesis in cornea which have a detrimental effect to vision and even lead to blindness. Bone morphogenetic protein 4 (BMP4) regulates a variety of biological processes, which is closely relevant to the regulation of corneal epithelium and angiogenesis. Herein, we aimed to evaluate the effect of BMP4 on corneal neovascularization (CNV), corneal lymphangiogenesis (CL), corneal epithelial repair, and the role of BMP4/Smad pathway in these processes. METHODS: We used MTT assay to determine the optimal concentration of BMP4. The suture method was performed to induce rat CNV and CL. We used ink perfusion and HE staining to visualize the morphological change of CNV, and utilized RT-qPCR and ELISA to investigate the expression of angiogenic factors and lymphangiogenic factors. The effects of BMP4 and anti-VEGF antibody on migration, proliferation and adhesion of corneal epithelium were determined by scratch test, MTT assay and cell adhesion test. RESULTS: BMP4 significantly inhibited CNV and possibly CL. Topical BMP4 resulted in increased expression of endogenous BMP4, and decreased expression of angiogenic factors and lymphangiogenic factors. Compared with anti-VEGF antibody, BMP4 enhanced corneal epithelium migration, proliferation and adhesion, which facilitated corneal epithelial injury repair. Simultaneously, these processes could be regulated by BMP4/Smad pathway. CONCLUSIONS: Our results demonstrated unreported effects of BMP4 on CNV, CL, and corneal epithelial repair, suggesting that BMP4 may represent a potential therapeutic target in corneal injury repair.

Recombinant human bone morphogenetic protein-4 enhances tendon-to-bone attachment healing in a murine model of rotator cuff tear.

BACKGROUND: Injuries of tendon-to-bone attachments (TBA) are common clinical challenges. Bone morphogenetic protein-4 (BMP-4) is potent in chondrogenesis. However, studies focusing on the influence of BMP-4 on the healing of TBA are scarce. Thus, this study's objective was to explore the effect of BMP-4 on the healing of TBA in a murine model of rotator cuff tear. METHODS: An injury model of the supraspinatus tendon (SST) insertion was established on a total of 120 mature C57 black (BL)/6 mice (12 weeks old), who were then randomly allocated into 3 groups: BMP-4, noggin (an inhibitor of all BMP activities), and control, At weeks 2 and 4 after surgery, the supraspinatus tendon-humerus complexes (SSTHC) were harvested for microradiographic, histologic, immunofluorescent, and biomechanical evaluations. RESULTS: Radiographic data showed that BMP-4 was able to improve the quality of subchondral bone, manifested as higher bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and lower trabecular spacing (Tb.Sp). Histologically, the BMP-4 group at week-2 and -4 showed a better TBA healing interface, characterized by better organizational integration and remodeling, thicker fibrocartilage layer, and more fibrocartilage cells. Immunofluorescence evaluation demonstrated that the number of SOX 9 positive cells in the BMP-4 group was significantly more than that in the control or noggin groups at postoperative weeks 2 and 4 (P<0.05 for all). Mechanical testing results at postoperative weeks 4 demonstrated the failure load, and stiffness in the BMP-4 group were significantly higher (P<0.05 for both), while in the noggin group were significantly lower (P<0.05 for both), compared to the control group. CONCLUSIONS: The BMP-4 might enhance TBA healing by promoting the regeneration of fibrocartilaginous enthesis and mineralization, while this process was inhibited by noggin. Thus, BMP-4 may be a potential therapy to augment TBA healing and finally lead to more rapid rehabilitation and reduce recurrent injury risk.

DYRK1A Negatively Regulates CDK5-SOX2 Pathway and Self-Renewal of Glioblastoma Stem Cells.

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.

Matrix Gla protein maintains normal and malignant hematopoietic progenitor cells by interacting with bone morphogenetic protein-4.

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.

Sequential stimulation with different concentrations of BMP4 promotes the differentiation of human embryonic stem cells into dental epithelium with potential for tooth formation.

BACKGROUND: Tooth loss caused by caries or injuries has a negative effect on human health; thus, it is important to develop a reliable method of tooth regeneration. Research on tooth regeneration has mainly focused on mouse pluripotent stem cells, mouse embryonic stem cells, and adult stem cells from various sources in mice, whereas little has examined the differentiation of human embryonic stem (hES) cells into dental epithelium (DE) and odontogenic potential in vivo. METHODS: In this study, we induced hES cells to differentiate into dental epithelium using different concentrations of bone morphogenetic protein 4 (BMP4). With 1 pM BMP4, the hES cells differentiated into oral ectoderm (OE). These cells were then stimulated with 30 pM BMP4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence showed the differentiation of OE and DE. The DE generated was mixed with embryonic day 14.5 mouse dental mesenchyme (DM) and transplanted into the renal capsules of nude mice. Thirty days later, the resulting tooth-like structures were examined by micro-computed tomography and hematoxylin and eosin staining. RESULTS: After 4 days of 1 pM BMP4 stimulation, Pitx1-positive OE formed. qRT-PCR and immunofluorescence revealed that induction with 30 pM BMP4 for 2 days caused the OE to differentiate into Pitx2/Dlx2/AMBN-positive DE-like cells. These cells also expressed ß-catenin and p-Smad1/5/8, which are key proteins in the Wnt/ß-catenin and Bmp signaling pathways, respectively. Thirty days after in vivo transplantation, six teeth with enamel and dentin had formed on the kidney. CONCLUSIONS: These results show that hES cells differentiated into DE after sequential stimulation with different concentrations of BMP4, and the DE thus generated showed odontogenic potential.

Increased levels of miR-124 in human dental pulp stem cells alter the expression of neural markers.

Auditory neuropathy is the particular form of deafness in humans which cannot be treated by replacement therapy. Human dental pulp stem cells (hDPSCs) are derived from an ectomesenchymal neural crest cell population. Therefore, they possess a promising capacity for neuronal differentiation and repair. miR-124, a key regulator of neuronal development in the inner ear, is expressed at high levels in auditory and vestibular neurons. Here, we evaluated the possible effect of miR-124 in alteration of neural protein markers expression. Using quantitative reverse transcription-PCR (qRT-PCR) analyses and immunofluorescence staining, we studied the expression patterns of neural progenitor markers (Nestin, NOTCH1, and SOX2) and neural markers (ß-tubulin III, GATA-3, and peripherin) upon transfection of hDPSCs with miR-124. The qRT-PCR results showed that Nestin was upregulated 6 h post-transfection. In contrast, Nestin expression exhibited a decreasing trend 24 h and 48 h post-transfection. Higher levels of ß-tubulin III, 6 h and 16 h post transfection in RNA level as compared with control cells, were determined in transfected DPSCs. However, ß-tubulin-III expression decreased 48 h post-transfection. The immunoflourescence results indicated that transfection of hDPSCs with miR-124, only affected Nestin among the studied neural progenitor and neural marker expression in protein level.

Improving recombinant bone morphogenetic protein-4 (BMP-4) production by autoregulatory feedback loop removal using BMP receptor-knockout CHO cell lines.

A Chinese hamster ovary (CHO) cell line producing recombinant human bone morphogenetic protein-4 (rhBMP-4) (CHO-BMP-4), which expresses essential components of BMP signal transduction, underwent autocrine BMP-4 signaling. RNA seq analysis on CHO host cells (DG44) treated with rhBMP-4 (20 µg/mL) suggested that rhBMP-4 induced signaling in CHO cells could be a critical factor in limiting rhBMP-4 production and should be removed to improve rhBMP-4 production in recombinant CHO (rCHO) cells. The inhibition of autocrine BMP signaling in CHO-BMP-4 cells by the addition of LDN-193189, a chemical inhibitor of BMP receptor type I, significantly increased the mRNA expression levels of rhBMP-4. To establish BMP signaling-free host cells, a BMP receptor, the BMPRIA or BMPRII gene in DG44 cells, was knocked out using CRISPR/Cas9 gene-editing technology. Using three different knockout (KO) host cell lines as well as a DG44 wild-type (wt) cell line, rCHO cell clones producing rhBMP-4 were generated by a stepwise selection with increasing methotrexate concentrations. KO-derived clones showed a significantly higher maximum rhBMP-4 concentration than wt-derived clones in both batch and fed-batch cultures. Unlike wt-derived clones, KO-derived cell clones were able to produce higher amounts of hBMP-4 transcripts and proteins in the stationary phase of growth and did not experience growth inhibition induced by rhBMP-4. The mean maximum rhBMP-4 concentration of KO host-derived clones was approximately 2.4-fold higher than that of wt-derived clones (P < 0.05). Taken together, the disruption of BMP signaling in CHO cells by knocking out the BMP receptor significantly improved rhBMP-4 production.

Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells.

Dental pulp cells (DPCs) are valuable cell source for dental regeneration, albeit their application is restricted by limited pluripotency due to current culture condition. Mouse embryonic fibroblasts (MEFs) are served as feeder layer to maintain undifferentiated state of iPSCs and ESCs with long-term in vitro culture. Bone morphogenetic protein 4 (BMP4) plays an important role in the regulation of undifferentiated state and lineage commitment of cells through modulation of microenvironment. However, so far little was known how micro environment affect the multipotency of dental derived cells. To demonstrate the effect of optimized culture condition on multipotency of DPCs, cell proliferation and senescence of DPCs with MEF and/or rhBMP4-CM were examined by CCK8, telomerase activity and flow cytometry. Multilineage differentiation was detected by immunofluorescent staining, Real-time PCR and western blot. Expression of BMP4/NFATc1/LIF in the co-culture medium was evaluated by ELISA and expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs was detected by Real-time PCR. NFATc1 inhibitor INCA-6 was applied to DPCs with MEF and/or rhBMP4-CM, expression of NFATc1/Oct-4/Sox2/c-Myc was examined by Realtime PCR and western blot. Our results demonstrated that DPCs cultured with MEF and/or rhBMP4-CM showed increased cell proliferation, telomerase rate and multilineage differentiation capability. MEF-CM enhanced expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs through secretion of BMP4/NFATc1 in the culture medium. INCA-6 effectively restrained the MEF/BMP4-CM induced upregulation of Oct-4/Sox2/c-Myc/NFATc1 in DPCs. These resuts indicate that both MEF-CM and BMP4-CM provided similar efficient culture system to improve the multipotency of DPCs, which might contribute to the application of DPCs in dental regeneration.

Isoflurane post-conditioning down-regulates expression of aquaporin 4 in rats with cerebral ischemia/reperfusion injury and is possibly related to bone morphogenetic protein 4/Smad1/5/8 signaling pathway.

AIM: Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining. RESULTS: Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P<0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P<0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P<0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P<0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P>0.05), but neuronal density increased (P<0.05). CONCLUSION: Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway.

Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells.

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of EC50 of 2.93 ng/ml.

Gene-gene interactions in APOL1-associated nephropathy.

BACKGROUND: Two APOL1 nephropathy variants confer substantial risk for non-diabetic end-stage kidney disease (ESKD) in African Americans (AAs). Since not all genetically high-risk individuals develop ESKD, modifying factors likely contribute. Forty-two potentially interactive single nucleotide polymorphisms (SNPs) from a genome-wide association study in non-diabetic ESKD were tested for interaction with APOL1 to identify genes modifying risk for non-diabetic nephropathy. METHODS: SNPs were examined in an expanded sample of 1367 AA non-diabetic ESKD cases and 1504 AA non-nephropathy controls, with validation in an independent family-based cohort containing 608 first-degree relatives of index cases with non-diabetic ESKD. Logistic regression and mixed models were fitted to test for interaction effects with APOL1 on ESKD, estimated kidney function and albuminuria. RESULTS: Among ESKD samples, 14 of 42 SNPs demonstrated suggestive APOL1 interaction with P-values <0.05. After Bonferroni correction, significant interactions with APOL1 were seen with SNPs in podocin (rs16854341; NPHS2, P = 8.0 × 10(-4)), in SDCCAG8 (rs2802723; P = 5.0 × 10(-4)) and near BMP4 (rs8014363; P = 1.0 × 10(-3)); with trends for ENOX1 (rs9533534; P = 2.2 × 10(-3)) and near TRIB1 (rs4457349; P = 5.7 × 10(-3)). The minor allele in NPHS2 markedly changed the APOL1-ESKD association odds ratio (OR) from 7.03 to 1.76 (∼50% reduction in effect per copy of the minor allele), rs2802723 changed the OR from 5.1 to 10.5, and rs8014363 increased the OR from 4.8 to 9.5. NPHS2 (P = 0.05) and SDCCAG8 (P = 0.03) SNPs demonstrated APOL1 interaction with albuminuria in independent family-based samples. CONCLUSIONS: Variants in NPHS2, SDCCAG8 and near BMP4 appear to interact with APOL1 to modulate the risk for non-diabetic ESKD in AAs.