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.

Salvianolic acid B ameliorates vascular endothelial dysfunction through influencing a bone morphogenetic protein 4-ROS cycle in diabetic mice.

AIM: This study investigated the roles of bone morphogenetic protein-4 (BMP4) and ROS in diabetic endothelial dysfunction and explored whether Salvianolic acid B (Sal B) improved endothelial function by affecting BMP4-ROS in diabetic mice. MAIN METHODS: db/db mice were orally administrated with Sal B (10 mg/kg/day) for one week while db/m + mice were injected with adenoviral vectors delivering BMP4 (3 × 108 pfu) and then received one week-Sal B treatment. ROS levels were assayed by DHE staining. Protein expression and phosphorylation were evaluated by Western blot. Aortic rings were suspended in myograph for force measurement. Flow-mediated dilatations in the second-order mesenteric arteries were determined by pressure myograph. KEY FINDINGS: We first revealed the existence of a BMP4-ROS cycle in db/db mice, which stimulated p38 MAPK/JNK/caspase 3 and thus participated in endothelial dysfunction. One week-treatment or 24 h-incubation with Sal B disrupted the cycle, suppressed p38 MAPK/JNK/caspase 3 cascade, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Importantly, in vivo Sal B treatment also improved flow-mediated dilatation in db/db mouse second order mesenteric arteries. Furthermore, in vivo BMP4 overexpression induced oxidative stress, stimulated p38 MAPK/JNK/caspase 3, and impaired EDRs in db/m + mouse aortas, which were all reversed by Sal B. SIGNIFICANCE: The present study demonstrates that Sal B ameliorates endothelial dysfunction through breaking the BMP4-ROS cycle and subsequently inhibiting p38 MAPK/JNK/caspase 3 in diabetic mice and provides evidence for the additional new mechanism underlying the benefit of Sal B against diabetic vasculopathy.

The postnatal pancreatic microenvironment guides ß cell maturation through BMP4 production.

Glucose homeostasis depends on regulated insulin secretion from pancreatic ß cells, which acquire their mature phenotype postnatally. The functional maturation of ß cells is regulated by a combination of cell-autonomous and exogenous factors; the identity of the latter is mostly unknown. Here, we identify BMP4 as a critical component through which the pancreatic microenvironment regulates ß cell function. By combining transgenic mouse models and human iPSCs, we show that BMP4 promotes the expression of core ß cell genes and is required for proper insulin production and secretion. We identified pericytes as the primary pancreatic source of BMP4, which start producing this ligand midway through the postnatal period, at the age ß cells mature. Overall, our findings show that the islet niche directly promotes ß cell functional maturation through the timely production of BMP4. Our study highlights the need to recapitulate the physiological postnatal islet niche for generating fully functional stem-cell-derived ß cells for cell replacement therapy for diabetes.

NLRP7 plays a functional role in regulating BMP4 signaling during differentiation of patient-derived trophoblasts.

Complete hydatidiform mole (HM) is a gestational trophoblastic disease resulting in hyperproliferation of trophoblast cells and absence of embryo development. Mutations in the maternal-effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts harboring NLRP7 mutations. Using whole transcriptome profiling during trophoblast differentiation, we showed that impaired NLRP7 expression results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers, and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient-derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators.

Continuous psychosocial stress stimulates BMP signaling in dorsal hippocampus concomitant with anxiety-like behavior associated with differential modulation of cell proliferation and neurogenesis.

Bone morphogenetic protein (BMP) signaling in the hippocampus regulates psychiatric behaviors and hippocampal neurogenesis in non-stress conditions; however, stress-induced changes in hippocampal BMP signaling have not yet been reported. Therefore, we sought to examine whether psychosocial stress, which induces psychiatric symptoms, affects hippocampal BMP signaling. A total of 32 male Sprague-Dawley rats were exposed to a psychosocial stress using a Resident/Intruder paradigm for ten consecutive days. Subsequently, rats were subjected to a battery of behavioral tests (novelty-suppressed feeding test, sucrose preference test, and forced swimming test) for the evaluation of adult neurogenesis and activity of BMP signaling in the dorsal and ventral hippocampus. Repeated social defeat promoted anxiety-like behaviors, but neither anhedonia nor behavioral despair. Socially defeated rats exhibited an increase in the number of Ki-67-positive cells, decrease in the number of doublecortin (DCX)-positive cells, and decrease only in the dorsal hippocampus of the ratio of DCX-positive to Ki-67-positive cells, a proxy for newly-born cell maturation speed and survival. In contrast, no differences were observed in the number of 5-Bromo-2'-deoxyuridine (BrdU)-positive cells, indicating survival of newly-born cells both in the dorsal and ventral hippocampus. Furthermore, psychosocial stress significantly increased the BMP-4 and phosphorylated Smad1/5/9 expression levels specifically in the dorsal hippocampus. Our findings suggest that repeated psychosocial stress activates BMP signaling and differently affects cell proliferation and neurogenesis exclusively in the dorsal hippocampus, potentially exacerbating anxiety-related symptoms. Targeting BMP signaling is a potential therapeutic strategy for psychiatric disorders.

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

Acute Lymphoblastic Leukaemia Cells Impair Dendritic Cell and Macrophage Differentiation: Role of BMP4.

Dendritic cells and macrophages are common components of the tumour immune microenvironment and can contribute to immune suppression in both solid and haematological cancers. The Bone Morphogenetic Protein (BMP) pathway has been reported to be involved in cancer, and more recently in leukaemia development and progression. In the present study, we analyse whether acute lymphoblastic leukaemia (ALL) cells can affect the differentiation of dendritic cells and macrophages and the involvement of BMP pathway in the process. We show that ALL cells produce BMP4 and that conditioned media from ALL cells promote the generation of dendritic cells with immunosuppressive features and skew M1-like macrophage polarization towards a less pro-inflammatory phenotype. Likewise, BMP4 overexpression in ALL cells potentiates their ability to induce immunosuppressive dendritic cells and favours the generation of M2-like macrophages with pro-tumoral features. These results suggest that BMP4 is in part responsible for the alterations in dendritic cell and macrophage differentiation produced by ALL cells.

BMP4 patterns Smad activity and generates stereotyped cell fate organization in spinal organoids.

Bone morphogenetic proteins (BMPs) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity in vivo, whether this is sufficient for pattern formation in vitro is unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cell-derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types, and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species-specific tempo of neural differentiation. Together, this study describes efficient methods for generating patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.

The role of the BMP4/Smad1 signaling pathway in mesangial cell proliferation: A possible mechanism of diabetic nephropathy.

AIMS: This study explored the role of the BMP4/Smad1 signaling pathway in mesangial matrix expansion during the process of diabetic nephropathy. MAIN METHODS: Diabetic rats were induced by high-fat feeding followed by an intraperitoneal injection of streptozotocin. Glomerular lesions were examined. Immunohistochemical analysis was performed in order to identify BMP4/Smad1 signaling proteins (BMP4, ALK3, and Smad1) and mesangial ECM proteins (Col1 and Col4) in kidney tissue. Cell proliferation and the expression of BMP4, Smad1 and Col4 were determined in cultured mesangial cells exposed to high glucose. The specific regulatory role of BMP4 was evaluated by detecting BMP4/Smad1 signaling pathway proteins and mesangial ECM proteins after blocking BMP4 both at the gene and protein levels. KEY FINDINGS: Rats with DN exhibited mesangial expansion and a thickened glomerular basement membrane. Immunohistochemical analysis of glomeruli showed increased expression of BMP4, Smad1, ALK3, Col1, and Col4 but less expression of MMP9 than observed in controls. High glucose induced slight proliferation of cultured rat mesangial cells after 48 h of incubation but there was no significant different from the control (p > 0.05). High glucose activated the BMP4/Smad1 signaling pathway and stimulated Col4 expression in mesangial cells. Both silencing of the bmp4 gene (with siRNA) and blocking BMP4 protein signaling (with the BMP4 protein antagonist Noggin) reduced the expression of ALK3, Smad1, Col4, and Col1 in high glucose-stimulated mesangial cells. SIGNIFICANCE: The BMP4/Smad1 signaling pathway is crucial to the progression of mesangial expansion, and suppressing this signaling pathway may present a novel therapeutic strategy for DN.

Hedgehog signaling patterns the oral-aboral axis of the mandibular arch.

Development of vertebrate jaws involves patterning neural crest-derived mesenchyme cells into distinct subpopulations along the proximal-distal and oral-aboral axes. Although the molecular mechanisms patterning the proximal-distal axis have been well studied, little is known regarding the mechanisms patterning the oral-aboral axis. Using unbiased single-cell RNA-seq analysis followed by in situ analysis of gene expression profiles, we show that Shh and Bmp4 signaling pathways are activated in a complementary pattern along the oral-aboral axis in mouse embryonic mandibular arch. Tissue-specific inactivation of hedgehog signaling in neural crest-derived mandibular mesenchyme led to expansion of BMP signaling activity to throughout the oral-aboral axis of the distal mandibular arch and subsequently duplication of dentary bone in the oral side of the mandible at the expense of tongue formation. Further studies indicate that hedgehog signaling acts through the Foxf1/2 transcription factors to specify the oral fate and pattern the oral-aboral axis of the mandibular mesenchyme.

Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens.

BACKGROUND: The tissue-specific transcriptional programs during normal development require tight control by distal cis-regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence-specific DNA-binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T-cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown. RESULTS: Here we describe the identification of a novel bipartite Gata3 lens-specific enhancer located ∼18 kb upstream from its transcriptional start site. We also found that a 5-kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP-1, Ets-, and Smad1/5-binding sites as well as binding sites for lens-associated DNA-binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2. CONCLUSIONS: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up-regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186-1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

The homeobox transcription factor MSX2 partially mediates the effects of bone morphogenetic protein 4 (BMP4) on somatic cell reprogramming.

Bone morphogenetic proteins (BMPs) induce mesenchymal-epithelial transition (MET) and enhance the generation of induced pluripotent stem cells (iPSCs). However, BMPs are also signaling molecules critical for arresting reprogramming in the pre-iPSC state. In this study, using mouse embryonic fibroblasts, we found that the time- and concentration-dependent effects of BMPs on reprogramming are mediated by Msh homeobox 2 (MSX2), a homeobox-containing transcription factor. BMPs up-regulated Msx2 by activating SMAD1/5, and MSX2 then directly bound to the promoters and up-regulated the expression of the cadherin 1 (Cdh1, also known as E-cadherin), GATA-binding protein 3 (Gata3), and Nanog genes. Cdh1 contributed to BMP4- and MSX2-induced MET and subsequently promoted reprogramming. On the other hand, GATA3 promoted reprogramming, possibly by up-regulating Spalt-like transcription factor 4 (SALL4) expression. As key transcriptional factors in maintaining pluripotency, up-regulation of SALL4 and NANOG enhanced reprogramming. Moreover, the ability of MSX2 to up-regulate Cdh1, Gata3, Nanog, and Sall4 was further potentiated in the presence of Krüppel-like factor 4 (KLF4). However, MSX2 did not mediate the effects of BMP4 signaling on activation of the microRNAs miR-205 and miR-200 or the inhibitory effects that arrested reprogramming in the pre-iPSC state. In conclusion, MSX2 partially mediates the effects of BMP4 signaling during reprogramming, improving our understanding of the role of BMP signaling in MET and of the connection between cell lineage specifiers such as MSX2 and GATA3 and pluripotency.

Physical-chemical mechanisms of pattern formation during gastrulation.

Gastrulation is a fundamental phase during the biological development of most animals when a single layer of identical embryo cells is transformed into a three-layer structure, from which the organs start to develop. Despite a remarkable progress in quantifying the gastrulation processes, molecular mechanisms of these processes remain not well understood. Here we theoretically investigate early spatial patterning in a geometrically confined colony of embryonic stem cells. Using a reaction-diffusion model, a role of Bone-Morphogenetic Protein 4 (BMP4) signaling pathway in gastrulation is specifically analyzed. Our results show that for slow diffusion rates of BMP4 molecules, a new length scale appears, which is independent of the size of the system. This length scale separates the central region of the colony with uniform low concentrations of BMP molecules from the region near the colony edge where the concentration of signaling molecules is elevated. The roles of different components of the signaling pathway are also explained. Theoretical results are consistent with recent in vitro experiments, providing microscopic explanations for some features of early embryonic spatial patterning. Physical-chemical mechanisms of these processes are discussed.

[Progress in the study on bone morphogenetic protein 2/4 in central nervous system].

As a member of transforming growth factor ß (TGF-ß) family, bone morphogenetic proteins (BMPs) are multi-functional factors and play critical roles in heart, cartilage, neural development and postnatal bone formation. It has been demonstrated that among the family, BMP2/4 have been reported to be especially important for the developmental and maturation of central nervous system (CNS). It has different, even opposite functions, in certain given circumstances, which could be a potential risk for BMPs' clinical use.

The role of bone morphogenetic proteins 2 and 4 in mouse dentinogenesis.

OBJECTIVE: The bone morphogenetic proteins (BMPs) play crucial roles in tooth development. However, several BMPs retain expression in the dentin of the fully patterned and differentiated tooth. We hypothesized that BMP signaling therefore plays a role in the function of the differentiated odontoblast, the job of which is to lay down and mineralize the dentin matrix. DESIGN: We generated mice deficient in Bmp2 and 4 using a dentin matrix protein 1 (Dmp1) promoter-driven cre recombinase that was expressed in differentiated odontoblasts. RESULTS: The first and second molars of these Bmp2 and Bmp4 double conditional knockout (DcKO) mice displayed reduced dentin and enlarged pulp chambers compared to cre-negative littermate controls. DcKO mouse dentin in first molars was characterized by small, disorganized dentinal fibers, a wider predentin layer, and reduced expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). DcKO mouse odontoblasts demonstrated increased type I collagen mRNA production, indicating that the loss of BMP signaling altered the rate of collagen gene expression in these cells. Bmp2 and Bmp4 single Dmp1-cre knockout mice displayed no discernable dentin phenotype. CONCLUSIONS: These data demonstrate that BMP signaling in differentiated odontoblasts is necessary for proper dentin production in mature teeth.

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.

A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning.

How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior.

Endothelial BMP4 Regulates Leukocyte Diapedesis and Promotes Inflammation.

Leukocyte recruitment is a fundamental event in the response of the innate immune system to injury. This process is promoted in part by the opening of endothelial cell adherens junctions that allows leukocyte extravasation through gaps between adjacent endothelial cells. VE-cadherin is a key component of endothelial cell adherens junctions and a negative regulator of leukocyte emigration. Accumulating evidence implicates bone morphogenetic protein (BMP) 4 as a critical regulator in vascular biology, but its role in leukocyte extravasation in vitro and in vivo has not been investigated so far. To assess the impact of BMP4 on leukocyte emigration in vivo, we used the thioglycollate-induced peritonitis model. C57BL/6 mice were intraperitoneally (i.p.) injected with recombinant BMP4 in addition to thioglycollate. Compared to solvent-treated controls, we observed higher accumulation of leukocytes in the peritoneal lavage of BMP4-treated mice indicating that BMP4 promotes leukocyte diapedesis into the inflamed peritoneal cavity. Endothelial cell-specific deletion of BMP4 in mice markedly diminished leukocyte diapedesis following thioglycollate administration suggesting that endothelial BMP4 is required for leukocyte recruitment. Consistent with these in vivo results, transwell migration assays with human umbilical vein endothelial cells (HUVECs) in vitro revealed that recombinant BMP4 enhanced leukocyte transmigration through the endothelial monolayer. Conversely, silencing of endothelial BMP4 by siRNA dampened leukocyte diapedesis in vitro. Mechanistic studies showed that loss of BMP4 improved endothelial junction stability by upregulation of VE-cadherin expression in vitro and in vivo. Vice versa, treatment of HUVECs with recombinant BMP4 decreased expression of VE-cadherin and impaired endothelial junction stability shown by Western blotting and immunocytochemistry. Finally, severe endothelial damage in HUVECs in response to serum of patients collected 24 h after survived cardiac arrest was accompanied by increase in leukocyte migration in transwell assays and activation of the BMP pathway most probably by upregulation of endothelial BMP4 RNA and protein expression. Collectively, the present study provides novel evidence that endothelial BMP4 controls leukocyte recruitment through a VE-cadherin-dependent mechanism and that BMP4-induced inflammation might be involved in the pathogenesis of endothelial cell damage following successful resuscitation after cardiac arrest.

Bone morphogenetic protein 4 promotes the survival and preserves the structure of flow-sorted Bhlhb5+ cochlear spiral ganglion neurons in vitro.

SGNs are the primary auditory neurons, and damage or loss of SGNs leads to sensorineural hearing loss. BMP4 is a growth factor that belongs to the TGF-ß superfamily and has been shown to play a key role during development, but little is known about its effect on postnatal cochlear SGNs in mice. In this study, we used the P3 Bhlhb5-cre/tdTomato transgenic mouse model and FACS to isolate a pure population of Bhlhb5+ SGNs. We found that BMP4 significantly promoted SGN survival after 7 days of culture. We observed fewer apoptotic cells and decreased expression of pro-apoptotic marker genes after BMP4 treatment. We also found that BMP4 promoted monopolar neurite outgrowth of isolated SGNs, and high concentrations of BMP4 preserved the number and the length of neurites in the explant culture of the modiolus harboring the SGNs. We showed that high concentration of BMP4 enhanced neurite growth as determined by the higher average number of filopodia and the larger area of the growth cone. Finally, we found that high concentrations of BMP4 significantly elevated the synapse density of SGNs in explant culture. Thus, our findings suggest that BMP4 has the potential to promote the survival and preserve the structure of SGNs.

Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells.

The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells.