Bone Morphogenetic Protein Inhibitor Research Articles

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Abstract 151: BMP signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

Abstract While retinoic acid (RA) has been successfully used for leukemia treatment for decades, the attempt to treat solid tumors with RA remains challenging, with less than 10% of neuroblastoma (NB) patients achieving complete remission when treated with RA alone. It has been believed the anti-cancer activity of retinoids is due to retinoid-induced terminal differentiation, however, what determines cell response or whether differentiation is the main effect of RA has never been fully understood. To better understand RA’s activity, we conducted genome-wide CRISPR modifier screens in RA-treated hyper-sensitive NB cell lines. Surprisingly, we found that, in these cells, RA primarily decreased cell viability via apoptosis or senescence, rather than differentiation—activities in which the CRISPR screens strongly implicated bone morphogenetic protein (BMP) signaling. BMP activation promoted apoptosis/senescence and sensitized cells to RA. Conversely, BMP inhibitors and SMAD9 (a critical transcription factor of the BMP pathway) knockout enhanced RA’s ability to induce differentiation but reduced cell sensitivity to RA. Our ChIP-seq and RNA-seq data showed these behaviors were mediated by the coordinated gene regulatory actions of RARA and BMP-family SMAD transcription factors. Furthermore, in a panel of 19 cell lines we screened with RA, we assessed the correlations between RA IC50 and the expression of all (~20,000) genes (publicly available data from the GDSC and Depmap). Remarkably, SMAD9 was the number 1 and number 10 most correlated gene, respectively, with RA IC50 (ranked by Pearson correlation coefficient; GDSC R = -0.92, P = 4.2 × 10−6; Depmap R = -0.81, P = 4.8 × 10−4). We also performed comprehensive large-scale drug screens in these cell lines with combinations of RA and a BMP activator FK506. RA exhibited a synergistic effect with FK506 in all the NB cell lines, very strikingly in some cell lines. All these data suggest BMP signaling is generally required for RA sensitivity and BMP activators are promising candidates in combination with RA to treat NB. Using published bulk and single cell RNA-seq data from NB patient samples, we found BMP signaling activity is relatively low in primary tumors, but well maintained in disseminated NB cells derived from bone marrow. This explains why RA is clinically used as a maintenance therapy and can only successfully treat the minimal residual disease and suggests that tumor microenvironment is a critical factor in determining cell response to RA. Overall, our study revealed that BMP controls NB cell fate and sensitivity in RA treatment. Notably, interactions between BMP signaling and RA are well established in developmental biology, where BMP signaling can tip cell fate decisions between differentiation, apoptosis, and senescence upon exposure to endogenous RA. Our data suggest that this developmental process can be maintained in NB cells, unveiling a novel mode of anti-neoplastic action. Citation Format: Min Pan, Yinwen Zhang, William C. Wright, Hyeong-Min Lee, Richard H. Chapple, Xueying Liu, Jonathan Low, Duane Currier, Allister J. Loughran, Dyer A. Dyer, Shondra M. Pruett, Burgess Freeman, Taosheng Chen, Brian J. Abraham, Elizabeth Stewart, John Easton, Paul Geeleher. BMP signaling determines neuroblastoma cell fate and sensitivity to retinoic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 151.

Grainyhead-like 2 interacts with noggin to regulate tissue fusion in mouse.

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2-/-;Nog+/- embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2-/- mice.

Paraspeckle protein NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 transcription

Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.

A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-β and BMP signaling in the Fsh-mediated spermatogonial fate

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-β superfamily. On the other hand, the specific roles of TGF-β and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-β (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-β or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-β inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-β and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-β and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-β subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-β (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

Myo/Nog Cells Increase in Response to Elevated Intraocular Pressure and Mitigate Ganglion Cell Death in a Mouse Model of Glaucoma

Glaucoma is one of the leading causes of blindness worldwide. Decreased aqueous humor drainage causes an increase in intraocular pressure (IOP), which in turn damages the ganglion cells of the retina and optic nerve. A mouse model of glaucoma was used to examine the behavior of Myo/Nog (M/N) cells, which were previously shown to respond to cataract surgery and retinopathy induced by hypoxia, light damage, and intravitreal injection of human retinal pigment epithelial cells. M/N cells express the skeletal-muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin, and brain-specific angiogenesis inhibitor 1 (BAI1). Glaucoma was induced by injecting microbeads into the anterior chamber (AC) of the right eye to obstruct the flow of aqueous humor into the trabecular meshwork. IOP was elevated within three days of addition of microbeads. Loss of retinal ganglion cells (RGCs) and thinning of the ganglion cell layer–nerve fiber layer (GCL-NFL) was observed in tissue sections by day 32. The injection of microbeads resulted in an increase in BAI1-positive (+) M/N cells in the trabecular meshwork, ciliary body, canal of Schlemm, cornea, and ganglion cell layer (GCL). M/N cells ingested microbeads. The effect of further increasing the population of M/N cells on IOP and RGC loss was determined by injecting BAI1+ cells isolated from the brain into the AC of both eyes. Exogenous M/N cells prelabeled with CellTracker™ Red were found in the same tissues as the endogenous population of M/N cells in eyes with and without elevated IOP. The addition of M/N cells did not significantly reduce IOP in bead-injected eyes. However, there were significantly more RGCs and the NFL was thicker in glaucomatous eyes with M/N cell supplementation than eyes injected with phosphate-buffered saline. The numbers of RGCs and NFL thickness were similar in glaucomatous and non-glaucomatous eyes after adding M/N cells. These results demonstrate that endogenous M/N cells respond to elevated IOP in the anterior and posterior segments in response to induction of glaucoma. M/N cells’ mitigation of RGC loss may reflect a neuroprotective effect within the retina, as opposed to a significant drop in IOP.

Characterization of erythroferrone oligomerization and its impact on BMP antagonism

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF related protein (CTRP) family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE, and that larger species are dispensable for BMP inhibition. Additionally, we used an in-silico approach to identify a helix, termed the ligand binding domain (LBD), that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the LBD is crucial for activity through luciferase assays and surface plasmon resonance (SPR) analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Characterization of erythroferrone structural domains relevant to its iron-regulatory function

Iron delivery to the plasma is closely coupled to erythropoiesis, the production of red blood cells, as this process consumes most of the circulating plasma iron. In response to hemorrhage and other erythropoietic stresses, increased erythropoietin stimulates the production of the hormone erythroferrone (ERFE) by erythrocyte precursors (erythroblasts) developing in erythropoietic tissues. ERFE acts on the liver to inhibit bone morphogenetic protein (BMP) signaling and thereby decrease hepcidin production. Decreased circulating hepcidin concentrations then allow the release of iron from stores and increase iron absorption from the diet. Guided by evolutionary analysis and Alphafold2 protein complex modeling, we used targeted ERFE mutations, deletions, and synthetic ERFE segments together with cell-based bioassays and surface plasmon resonance to probe the structural features required for bioactivity and BMP binding. We define the ERFE active domain and multiple structural features that act together to entrap BMP ligands. In particular, the hydrophobic helical segment 81 to 86 and specifically the highly conserved tryptophan W82 in the N-terminal region are essential for ERFE bioactivity and Alphafold2 modeling places W82 between two tryptophans in its ligands BMP2, BMP6, and the BMP2/6 heterodimer, an interaction similar to those that bind BMPs to their cognate receptors. Finally, we identify the cationic region 96-107 and the globular TNFα-like domain 186-354 as structural determinants of ERFE multimerization that increase the avidity of ERFE for BMP ligands. Collectively, our results provide further insight into the ERFE-mediated inhibition of BMP signaling in response to erythropoietic stress.

RAS‑stimulated release of exosomal miR‑494‑3p promotes the osteolytic bone metastasis of breast cancer cells.

RAS activation is a key determinant of breast cancer progression and metastasis. However, the role of the interaction among exosomes, RAS and microRNAs (miRNAs/miRs) in the osteolytic bone metastasis of breast cancer remains unclear. Therefore, the present study aimed to examine the role of activated RAS (KRAS, HRAS and NRAS) in the release of exosome‑mediated osteoclastogenic miRNAs and to elucidate their functional role in bone microenvironment remodeling invitro and invivo. Exosomes derived from RAS‑activated breast cancer cells promoted RANKL‑induced osteoclastogenesis; however, RAS inhibition abolished this effect. miR‑494‑3p, miR‑4508 and miR‑6869‑5p were identified as osteoclastogenic miRNAs in the exosomes secreted by RAS‑activated breast cancer cells. The levels of these osteoclastogenic miRNAs in the sera of patients with human epidermal growth factor receptor 2‑positive luminal breast cancer were significantly higher than those in the sera of patients with triple‑negative breast cancer. miR‑494‑3p exhibited both osteoclastogenic and anti‑osteoblastogenic activity. Treatment with a miR‑494‑3p inhibitor abolished the exosome‑mediated increase in RANKL‑induced osteoclastogenesis. Treatment with a miR‑494‑3p mimic enhanced RANKL‑induced osteoclast formation; however, treatment with its inhibitor suppressed this effect by targeting leucine‑rich repeat‑containing G‑protein coupled receptor 4 in osteoclast precursors. Furthermore, miR‑494‑3p inhibited bone morphogenetic protein 2‑induced osteoblast formation by targeting semaphorin 3A. In a mouse model, exosomes derived from breast cancer cells promoted osteolytic bone lesions; however, treatment with a miR‑494‑3p inhibitor significantly suppressed this effect. On the whole, the present study provides a novel mechanism, demonstrating that the RAS activation of breast cancer cells induces osteolytic bone metastasis by stimulating the exosome‑mediated transfer of osteoclastogenic miRNAs, including miR‑494‑3p to bone cells.

Myo/Nog Cells: The Jekylls and Hydes of the Lens.

Herein, we review a unique and versatile lineage composed of Myo/Nog cells that may be beneficial or detrimental depending on their environment and nature of the pathological stimuli they are exposed to. While we will focus on the lens, related Myo/Nog cell behaviors and functions in other tissues are integrated into the narrative of our research that spans over three decades, examines multiple species and progresses from early stages of embryonic development to aging adults. Myo/Nog cells were discovered in the embryonic epiblast by their co-expression of the skeletal muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin and brain-specific angiogenesis inhibitor 1. They were tracked from the epiblast into the developing lens, revealing heterogeneity of cell types within this structure. Depletion of Myo/Nog cells in the epiblast results in eye malformations arising from the absence of Noggin. In the adult lens, Myo/Nog cells are the source of myofibroblasts whose contractions produce wrinkles in the capsule. Eliminating this population within the rabbit lens during cataract surgery reduces posterior capsule opacification to below clinically significant levels. Parallels are drawn between the therapeutic potential of targeting Myo/Nog cells to prevent fibrotic disease in the lens and other ocular tissues.

Inhibition of the bone morphogenetic protein pathway suppresses tumor growth through downregulation of epidermal growth factor receptor in MEK/ERK-dependent colorectal cancer.

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, its role in colorectal cancer (CRC) is controversial, where it can act as context-dependent tumor promoter or tumor suppressor. Here we have found that CRC cells reside in a BMP-rich environment based on curation of two publicly available RNA-sequencing databases. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of select CRC organoids. Colorectal cancer organoids treated with LDN193189 showed a decrease in epidermal growth factor receptor, which was mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) expression. Among 18 molecularly characterized CRC organoids, suppression of growth by BMP inhibition correlated with induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, in CRC organoids, which are susceptible to growth suppression by LDN193189, simultaneous treatment with LDN193189 and trametinib, an FDA-approved MEK inhibitor, resulted in cooperative growth inhibition both in vitro and in vivo. Taken together, the simultaneous inhibition of BMP and MEK could be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders to this regimen.

Maternal RNA binding protein with multiple splicing 2 (RBPMS2) is involved in mouse blastocyst formation through the bone morphogenetic protein pathway

Research questionIs early embryo development in mice influenced by RNA binding protein with multiple splicing 2 (RBPMS2), a maternal factor that accumulates and is stored in the cytoplasm of mature oocytes? DesignThe expression patterns of RBPMS2 in mouse were analysed using quantitative real-time PCR (qRT PCR) and immunofluorescence staining. The effect of knockdown of RBPMS2 on embryo development was evaluated through a microinjection of specific morpholino or small interfering RNA. RNA sequencing was performed for mechanistic analysis. The interaction between RBPMS2 and the bone morphogenetic protein (BMP) pathway was studied using BMP inhibitor and activator. The effect on the localization of E-cadherin was determined by immunofluorescence staining. ResultsMaternal protein RBPMS2 is highly expressed in mouse oocytes, and knockdown of RBPMS2 inhibits embryo development from the morula to the blastocyst stage. Mechanistically, RNA sequencing showed that the differentially expressed genes were enriched in the transforming growth factor-β (TGF-β) signalling pathway. BMPs are members of the TGF-β superfamily of growth factors. It was found that the addition of BMP inhibitor to the culture medium led to a morula-stage arrest, similar to that seen in RBPMS2 knockdown embryos. This morula-stage arrest defect caused by RBPMS2 knockdown was partially rescued by BMP activator. Furthermore, the localization of E-cadherin to the membrane was impaired in response to a knockdown of RBPMS2 or inhibition of the BMP pathway. ConclusionThis study suggests that RBPMS2 activates the BMP pathway and thus influences the localization of E-cadherin, which is important for early mouse embryo development during blastocyst formation.

Clinicopathological relevance of stem cell marker growth and differentiation factor 3 in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the second leading cause of cancer-related deaths in Iran, often diagnosed in advanced stages with a poor prognosis. Growth and differentiation factor 3 (GDF3) is a member of the transforming growth factor-beta (TGF-β) superfamily. It acts as an inhibitor of bone morphogenetic proteins (BMPs) signaling pathway associated with pluripotent embryonic and cancer stem cells (CSCs) characteristics. Since its expression in ESCC has not yet been evaluated, the clinicopathological relevance of GDF3 expression was elucidated in ESCC patients. Expression of GDF3 in tumor tissues from 40 ESCC patients was compared to the related margin normal tissues by relatively comparative real-time polymerase chain reaction (PCR). Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. Likewise, the function of GDF3 in the differentiation and development of embryonic stem cells (ESCs) was also reviewed. GDF3 was significantly overexpressed in 17.5% of tumors and a significant correlation between GDF3 expression and the depth of tumor invasion was observed (P = 0.032). The results suggest that GDF3 expression is likely to have substantial roles in the progression and invasiveness behavior of ESCC. Having considered the importance of CSC markers identification and their exploitation in targeted cancer therapy, GDF3 may be introduced as a promising therapeutic target to inhibit the invasion of tumor cells in ESCC.

Abstract 2844: BMP inhibition overcomes chemoresistance of prostate cancer

Abstract Chemoresistance is a major therapeutic challenge to prostate cancer and its underlying molecular mechanism is poorly understood. Previously, it has been suggested that bone morphogenetic protein (BMP) signaling is down-regulated during the prostate cancer progression from the early androgen-sensitive stage to the metastatic castration-resistant stage. However, no literature reports are available for BMP signaling in the more advanced-chemoresistant prostate cancer. In this study, we found the expression levels of the BMP type I receptor members, Activin-like kinase-2 (ALK2) and Activin-like kinase-3 (ALK3), were significantly higher in the chemoresistant prostate cancer cells than those in the chemosensitive prostate cancer cells. In addition, the phospho-Smad1/5/9 proteins, the pivotal intracellular effectors of the BMP signaling, were notably elevated in the chemoresistant prostate cancer cells over the chemosensitive prostate cancer cells, indicating that BMP signaling is highly activated in the chemoresistant prostate cancer cells. We also found that BMP signaling inhibition with either DMH1 or the knockdown of ALK2/ALK3 sensitized chemoresistant prostate cancer cells to the chemotherapy drug docetaxel in a dose dependent manner. Our further study indicates that DMH1 suppressed the migration and invasion of chemoresistant prostate cancer cells in vitro, and attenuated chemoresistant prostate tumor growth in the mouse xenograft model in vivo. In addition, we showed that DMH1 disrupted the sphere formation in DU145-TxR and PC3-TxR cells, suppressed the expression of marker genes of the cancer stem cells (CSCs), and inhibited the efflux activity of multidrug resistance P-glycoprotein (P-gp1). In conclusion, our study demonstrates that BMP signaling is associated with prostate cancer chemoresistance and BMP signaling inhibition effectively overcomes the cancer chemoresistance potentially through the disruption of CSCs’ stemness and P-gp1 efflux activity. Citation Format: Jijun Hao, Chen Xie, Zhijun Wang, Yong Bar, Jose Aguilar, Austin Kyan, Li Zhing. BMP inhibition overcomes chemoresistance of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2844.

Smooth muscle contributes to the development and function of a layered intestinal stem cell niche.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The invivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

Intrinsic BMP inhibitor Gremlin regulates alveolar epithelial type II cell proliferation and differentiation

Type 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Bleomycin-induced lung injury upregulated Gremlin and showed a similar time course of biomarker expression comparable to the adenovirus model. TGF-β and IL-1β induced Gremlin expression in fibroblasts. Taken together, our findings implicate that Gremlin expression during lung injury leads to precisely timed inhibition of BMP signaling and activates AT2s, leading to alveolar repair.

Dual mechanism underlying failure of neural tube closure in the Zic2 mutant mouse.

Understanding the molecular mechanisms that lead to birth defects is an important step towards improved primary prevention. Mouse embryos homozygous for the Kumba (Ku) mutant allele of Zic2 develop severe spina bifida with complete lack of dorsolateral hinge points (DLHPs) in the neuroepithelium. Bone morphogenetic protein (BMP) signalling is over-activated in Zic2Ku/Ku embryos, and the BMP inhibitor dorsomorphin partially rescues neural tube closure in cultured embryos. RhoA signalling is also over-activated, with accumulation of actomyosin in the Zic2Ku/Ku neuroepithelium, and the myosin inhibitor Blebbistatin partially normalises neural tube closure. However, dorsomorphin and Blebbistatin differ in their effects at tissue and cellular levels: DLHP formation is rescued by dorsomorphin but not Blebbistatin, whereas abnormal accumulation of actomyosin is rescued by Blebbistatin but not dorsomorphin. These findings suggest a dual mechanism of spina bifida origin in Zic2Ku/Ku embryos: BMP-dependent formation of DLHPs is faulty, together with RhoA-dependent F-actin accumulation in the neuroepithelium. Hence, we identify a multi-pathway origin of spina bifida in a mammalian system that may provide a developmental basis for understanding the corresponding multifactorial human defects.

Rhodopsin-positive cell production by intravitreal injection of small molecule compounds in mouse models of retinal degeneration.

We aimed to verify whether the intravitreal injection of small molecule compounds alone can create photoreceptor cells in mouse models of retinal degeneration. Primary cultured mouse Müller cells were stimulated in vitro with combinations of candidate compounds and the rhodopsin expression was measured on day 7 using polymerase chain reaction and immunostaining. We used 6-week-old N-methyl-N-nitrosourea-treated and 4-week-old rd10 mice as representative in vivo models of retinal degeneration. The optimal combination of compounds selected via in vitro screening was injected into the vitreous and the changes in rhodopsin expression were investigated on day 7 using polymerase chain reaction and immunostaining. The origin of rhodopsin-positive cells was also analyzed via lineage tracing and the recovery of retinal function was assessed using electroretinography. The in vitro mRNA expression of rhodopsin in Müller cells increased 30-fold, and 25% of the Müller cells expressed rhodopsin protein 7 days after stimulation with a combination of 4 compounds: transforming growth factor-β inhibitor, bone morphogenetic protein inhibitor, glycogen synthase kinase 3 inhibitor, and γ-secretase inhibitor. The in vivo rhodopsin mRNA expression and the number of rhodopsin-positive cells in the outer retina were significantly increased on day 7 after the intravitreal injection of these 4 compounds in both N-methyl-N-nitrosourea-treated and rd10 mice. Lineage tracing in td-Tomato mice treated with N-methyl-N-nitrosourea suggested that the rhodopsin-positive cells originated from endogenous Müller cells, accompanied with the recovery of the rhodopsin-derived scotopic function. It was suggested that rhodopsin-positive cells generated by compound stimulation contributes to the recovery of retinal function impaired by degeneration.

Expression and Function of BMP and Activin Membrane-Bound Inhibitor (BAMBI) in Chronic Liver Diseases and Hepatocellular Carcinoma.

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) is a transmembrane pseudoreceptor structurally related to transforming growth factor (TGF)-β type 1 receptors (TGF-β1Rs). BAMBI lacks a kinase domain and functions as a TGF-β1R antagonist. Essential processes such as cell differentiation and proliferation are regulated by TGF-β1R signaling. TGF-β is the best-studied ligand of TGF-βRs and has an eminent role in inflammation and fibrogenesis. Liver fibrosis is the end stage of almost all chronic liver diseases, such as non-alcoholic fatty liver disease, and at the moment, there is no effective anti-fibrotic therapy available. Hepatic BAMBI is downregulated in rodent models of liver injury and in the fibrotic liver of patients, suggesting that low BAMBI has a role in liver fibrosis. Experimental evidence convincingly demonstrated that BAMBI overexpression is able to protect against liver fibrosis. Chronic liver diseases have a high risk of hepatocellular carcinoma (HCC), and BAMBI was shown to exert tumor-promoting as well as tumor-protective functions. This review article aims to summarize relevant studies on hepatic BAMBI expression and its role in chronic liver diseases and HCC.

Why Some Mice Are Smarter than Others: The Impact of Bone Morphogenetic Protein Signaling on Cognition.

Inbred mice (C57Bl/6) display wide variability in performance on hippocampal-dependent cognitive tasks. Examination of microdissected dentate gyrus (DG) after cognitive testing showed a highly significant negative correlation between levels of bone morphogenetic protein (BMP) signaling and recognition memory. Cognitive performance decline during the aging process, and the degree of cognitive decline is strongly correlated with aging-related increases in BMP signaling. Further, cognitive performance was impaired when the BMP inhibitor, noggin, was knocked down in the DG. Infusion of noggin into the lateral ventricles enhanced DG-dependent cognition while BMP4 infusion led to significant impairments. Embryonic overexpression of noggin resulted in lifelong enhancement of recognition and spatial memory while overexpression of BMP4 resulted in lifelong impairment, substantiating the importance of differences in BMP signaling in wild-type mice. These findings indicate that performance in DG-dependent cognitive tasks is largely determined by differences in levels BMP signaling in the dentate gyrus.