Downregulated BMP-Smad1/5/8 signaling causes emphysema via dysfunction of alveolar type II epithelial cells.

Bone morphogenetic protein (BMP)-Smad1/5/8 signaling plays a crucial regulatory role in lung development and adult lung homeostasis. However, it remains elusive whether BMP-Smad1/5/8 signaling is involved in the pathogenesis of emphysema. In this study, we downregulated BMP-Smad1/5/8 signaling by overexpressing its antagonist Noggin in adult mouse alveolar type II epithelial cells (AT2s), resulting in an emphysematous phenotype mimicking the typical pathological features of human emphysema, including distal airspace enlargement, pulmonary inflammation, extracellular matrix remodeling, and impaired lung function. Dysregulation of BMP-Smad1/5/8 signaling in AT2s leads to inflammatory destruction dominated by macrophage infiltration, associated with reduced secretion of surfactant proteins and inhibition of AT2 proliferation and differentiation. Reactivation of BMP-Smad1/5/8 signaling by genetics or chemotherapy significantly attenuated the morphology and pathophysiology of emphysema and improved the lung function in Noggin-overexpressing lungs. We also found that BMP-Smad1/5/8 signaling was downregulated in cigarette smoke-induced emphysema, and that enhancing its activity in AT2s prevented or even reversed emphysema in the mouse model. Our data suggest that BMP-Smad1/5/8 signaling, located at the top of the signaling cascade that regulates lung homeostasis, represents a key molecular regulator of alveolar stem cell secretory and regenerative function, and could serve as a potential target for future prevention and treatment of pulmonary emphysema. © 2023 The Pathological Society of Great Britain and Ireland.

BMP/Smad Pathway Is Involved in Lithium Carbonate-Induced Neural-Tube Defects in Mice and Neural Stem Cells.

Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li2CO3 treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li2CO3 at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li2CO3 or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li2CO3-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li2CO3 exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li2CO3 exposure via inositol deficiency.

Bmp4 Synexpression Gene, Sizzled, Transcription Is Collectively Modulated by Smad1 and Ventx1.1/Ventx2.1 in Early Xenopus Embryos.

Sizzled (Szl) is a secreted frizzled protein, having a sequence homology with the extracellular cysteine-rich domain (CRD) of the Wnt receptor, 'Frizzled'. Contrary to the other secreted frizzled like proteins (Sfrps), szl belongs to the bone morphogenetic protein 4 (Bmp4) synexpression group and is tightly coexpressed with Bmp4. What is not known is how the szl transcription achieves its Bmp4 synexpression pattern. To address the molecular details of szl transcription control, we cloned a promoter of size 1566 base pairs for szl (bps) from the Xenopus laevis genomic DNA. Luciferase and eGFP reporter gene results of this szl promoter (-1566 bp) in its activation and repression patterns by Bmp4/Smad1 and a dominant negative Bmp4 receptor (DNBR) were similar to those of the endogenous szl expression. Reporter gene assays and site-directed mutagenesis of the szl promoter mapped an active Bmp4/Smad1 response element (BRE) and a cis-acting element, which competitively share a direct binding site for Ventx1.1 and Ventx2.1 (a Ventx response element, VRE). Smad1 and ventx2.1 alone increased szl promoter activity; in addition, the binding of each protein component was enhanced with their coexpression. Interestingly, Ventx1.1 repressed this reporter gene activity; however, Ventx1.1 and Ventx2.1 together positively regulated the szl promoter activity. From our analysis, Ventx2.1 binding was enhanced by Ventx1.1, but Ventx1.1 inhibitory binding was inhibited by co-injection of Ventx2.1 for the VRE site. The inhibitory Ventx1.1 co-injection decreased Smad1 binding on the szl promoter. In a triple combination of overexpressed Smad1/Ventx1.1/Ventx2.1, the reduced binding of Smad1 from Ventx1.1 was recovered to that of the Smad1/Ventx2 combination. Collectively, this study provides evidence of Bmp4/Smad1 signaling for a primary immediate early response and its two oppositely behaving target transcription factors, Ventx1.1 and Ventx2.1, for a secondary response, as they together upregulate the szl promoter's activity to achieve szl expression in a Bmp4 synexpression manner.

Transmembrane anterior posterior transformation 1 regulates BMP signaling and modulates the protein stability of SMAD1/5.

The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2 cells was observed in an in vivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling.

BMP/SMAD1/5 Signaling in the Endometrial Epithelium Is Essential for Receptivity and Early Pregnancy.

The biological processes that control endometrial receptivity and embryo implantation are critical for the successful outcome of pregnancy. The endometrium is the complex inner lining of the uterine wall that is under the cyclical control of estrogen and progesterone and is a site of intimate contact between mother and blastocyst. The bone morphogenetic signaling (BMP) pathway is a highly conserved signaling pathway that controls key cellular processes throughout pregnancy and exerts intracellular effects via the SMAD1/5 transcription factors. To delineate the endometrial compartment-specific roles of BMP signaling, we generated mice with epithelial-specific conditional deletion of SMAD1/5 using Lactoferrin-icre (Smad1flox/flox;Smad5flox/flox;Lactoferrin-cre, "Smad1/5 cKO"). Histological analysis of the reproductive tracts showed that Smad1/5 cKO mice were developmentally normal and displayed no defects in glandular morphology. In fertility analyses, single SMAD1 or SMAD5 deletion had no effect on fertility; however, double-conditional deletion of SMAD1 and SMAD5 resulted in severe subfertility. Timed mating analyses revealed endometrial receptivity defects in the Smad1/5 cKO mice beginning at 3.5 days post coitum (dpc) that perturbed embryo implantation at 4.5 dpc, as demonstrated by the detection of unattached blastocysts in the uterus, decreased COX2 expression, and FOXO1 cytoplasmic mislocalization. We also found that defects that arose during peri-implantation adversely affected embryonic and decidual development at 5.5 and 6.5 dpc. Thus, uterine epithelial BMP/SMAD1/5 signaling is essential during early pregnancy and SMAD1/5 epithelial-specific deletion has detrimental effects on stromal cell decidualization and pregnancy development.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Adipose-derived mesenchymal stromal cell-derived exosomes promote tendon healing by activating both SMAD1/5/9 and SMAD2/3.

BACKGROUND: The use of adipose-derived mesenchymal stromal cell-derived exosomes (ADSC-Exos) may become a new therapeutic method in biomedicine owing to their important role in regenerative medicine. However, the role of ADSC-Exos in tendon repair has not yet been evaluated. Therefore, we aimed to clarify the healing effects of ADSC-Exos on tendon injury. METHODS: The adipose-derived mesenchymal stromal cells (ADSCs) and tendon stem cells (TSCs) were isolated from the subcutaneous fat and tendon tissues of Sprague-Dawley rats, respectively, and exosomes were isolated from ADSCs. The proliferation and migration of TSCs induced by ADSC-Exos were analyzed by EdU, cell scratch, and transwell assays. We used western blot to analyze the tenogenic differentiation of TSCs and the role of the SMAD signaling pathways. Then, we explored a new treatment method for tendon injury, combining exosome therapy with local targeting using a biohydrogel. Immunofluorescence and immunohistochemistry were used to detect the expression of inflammatory and tenogenic differentiation after tendon injury, respectively. The quality of tendon healing was evaluated by hematoxylin-eosin (H&E) staining and biomechanical testing. RESULTS: ADSC-Exos could be absorbed by TSCs and promoted the proliferation, migration, and tenogenic differentiation of these cells. This effect may have depended on the activation of the SMAD2/3 and SMAD1/5/9 pathways. Furthermore, ADSC-Exos inhibited the early inflammatory reaction and promoted tendon healing in vivo. CONCLUSIONS: Overall, we demonstrated that ADSC-Exos contributed to tendon regeneration and provided proof of concept of a new approach for treating tendon injuries.

RUNX1 regulates SMAD1 by transcriptionally activating the expression of USP9X, regulating the activation of hepatic stellate cells and liver fibrosis.

Liver fibrosis (LF) is a common pathological process with high morbidity and mortality. Runt-related transcription factor 1 (RUNX1) is a transcription factor that could cause nephropathy and renal fibrosis, but its role in LF is unclear. Therefore, this study aimed to investigate the role RUNX1 in LF. Briefly, hepatic fibrosis was detected by Sirius Red staining. Transcript levels were quantified by qPCR, and proteins were assessed by western blotting or immunofluorescence. Cell viability and cell migration were measured by CCK8 assays and wound healing assays, respectively. The binding of RUNX1 and ubiquitin-specific protease 9X (USP9X) promoter was validated by ChIP assays and luciferase report assays, while the binding of USP9X and SMAD1 was confirmed by co-immunoprecipitation (Co-IP). Our studies found that the expression of RUNX1 was upregulated in LF mice, and RUNX1 knockdown alleviated CCl4-induced LF. RUNX1 silencing reduced the viability and migration of HSCs. Besides, RUNX1, as a transcription factor, bound to the promoter of USP9X and regulated the expression of USP9X. USP9X is a deubiquitination enzyme and was found to be up-regulated in LF mice. USP9X silencing reduced the viability and migration of HSCs, thereby inhibiting LF. Further studies showed that USP9X could stabilize downstream Smad1 expression. Furthermore, we also found that RUNX1 regulated the expression of SMAD1 by transcriptionally activating the expression of USP9X, thereby regulating the activation of hepatic stellate cells and liver fibrosis.

MicroRNA-182 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Smad1.

The present study aimed to screen abnormally expressed microRNAs (miRs/miRNAs) in patients with postmenopausal osteoporosis (POP) and explore their mechanisms via functional verification. Bone marrow mesenchymal stem cells (BMSCs) were extracted from healthy controls and patients with POP. Differences in osteogenic differentiation and proliferation of human BMSCs were compared between the two groups using Cell Counting Kit-8 (CCK-8) assay and alizarin red staining. A rat model of POP was established. Compared with patients with POP, human BMSCs in healthy controls had significantly enhanced viability at 24, 36, 48 and 72 h. The results of alizarin red staining revealed that the deposition of calcium minerals in human BMSCs were significantly lower in patients with POP. Based on miRNA microarray and reverse transcription-quantitative polymerase chain reaction (PCR) results, the expression levels of miR-7010 and miR-467c decreased, while miR-132 and miR-182 expression increased in the human BMSCs of patients with POP. Alizarin red staining showed that miR-182 markedly suppressed the osteogenic differentiation of primary rat BMSCs in rats. Western blotting and immunofluorescence assay revealed that miR-182 inhibited the expression of osteogenesis markers runt-related transcription factor 2, osterix and actinin-associated LIM protein. The results of the luciferase reporter assay showed that Smad1 is the direct target of miR-182. In rat primary BMSCs, Smad1 overexpression abolished the inhibitory effect of miR-182 on osteogenesis, indicating that miR-182 inhibits osteogenic differentiation of primary rat BMSCs in rats by targeting Smad1. Finally, in vivo experimental results showed that the biomechanical characteristics of bone tissues in POP rats were significantly enhanced by miR-182 inhibition, while they were significantly weakened by miR-182 overexpression. MiR-182 inhibits osteogenic differentiation of rat BMSCs, thus aggravating POP in rats.

HOXD13 suppresses prostate cancer metastasis and BMP4-induced epithelial-mesenchymal transition by inhibiting SMAD1.

The HOX genes are a group of highly conserved Homeobox-containing genes that control the body plan organization during development. However, their contributions to tumorigenesis and tumor progression remain uncertain and controversial. Here we provided evidence of tumor-suppressive activity of HOXD13 in prostate cancer. HOXD13 depletion contributes to more aggressiveness of prostate cancer cells in vitro and in vivo. These effects were corroborated in a metastatic mice model, where we observed more bone metastatic lesions formed by prostate cancer cells with HOXD13 ablation. Mechanistically, HOXD13 prevents BMP4-induced epithelial-mesenchymal transition (EMT) by inhibiting mothers against decapentaplegic homolog 1 (SMAD1) transcription. Both bioinformation and our tissue microarray cohort data show that HOXD13 expression inversely correlated in advanced prostate cancer patient specimens. Our findings establish HOXD13 as a negative regulator of prostate cancer progression and metastasis by preventing BMP4/SMAD1 signaling, and potentially suggest new strategies for targeting metastatic prostate cancer.

Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/ß-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast-adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/ß-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

Lack of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Disturbs Callus Formation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally secreted signaling peptide and has important regulatory roles in the differentiation of the central nervous system and its absence results in disorders in femur development. PACAP has an important function in prevention of oxidative stress or mechanical stress in chondrogenesis but little is known about its function in bone regeneration. A new callus formation model was set to investigate its role in bone remodeling. Fracturing was 5 mm distal from the proximal articular surface of the tibia and the depth was 0.5 mm. Reproducibility of callus formation was investigated with CT 3, 7, and 21 days after the operation. Absence of PACAP did not alter the alkaline phosphatase (ALP) activation in PACAP KO healing process. In developing callus, the expression of collagen type I increased in wild-type (WT) and PACAP KO mice decreased to the end of healing process. Expression of the elements of BMP signaling was disturbed in the callus formation of PACAP KO mice, as bone morphogenic protein 4 (BMP4) and 6 showed an early reduction in bone regeneration. However, elevated Smad1 expression was demonstrated in PACAP KO mice. Our results indicate that PACAP KO mice show various signs of disturbed bone healing and suggest PACAP compensatory and fine tuning effects in proper bone regeneration.

Variants of SMAD1 gene increase the risk of colorectal cancer in the Bangladeshi population.

Colorectal cancer is the fourth most common type of malignancy worldwide that may develop due to the accumulation of several genetic variations. Different single nucleotide polymorphisms of SMAD1 gene are assumed to be linked with increased colorectal cancer risk. The current case-control study was conducted to verify the association of genetic polymorphisms of SMAD1 (rs11100883 and rs7661162) with colorectal cancer in the Bangladeshi population. This study was performed on 275 colorectal cancer patients and 300 healthy volunteers using polymerase chain reaction-restriction fragment length polymorphism method. The odds ratios were adjusted for age and sex with logistic regression analysis. In case of SMAD1 rs11100883 polymorphism, GA heterozygous genotype, GA + AA (dominant model), and minor allele "A" were significantly associated with colorectal cancer (adjusted odds ratio = 1.55, 95% confidence interval = 1.09-2.20, p = 0.014; adjusted odds ratio = 1.59, 95% confidence interval = 1.13-2.23, p = 0.008; and odds ratio = 1.35, 95% confidence interval = 1.06-1.73, p = 0.015, respectively) and the significance exists after the Bonferroni correction. Again, single nucleotide polymorphism rs7661162 showed significant association with an elevated colorectal cancer risk for AG heterozygous genotype, AG + GG (dominant model), AG versus AA + GG (overdominant model), and minor allele "G" (adjusted odds ratio = 1.78, 95% confidence interval = 1.24-2.56, p = 0.002; adjusted odds ratio = 1.68, 95% confidence interval = 1.18-2.39, p = 0.004; adjusted odds ratio = 1.76, 95% confidence interval = 1.23-2.53, p = 0.002; and odds ratio = 1.47, 95% confidence interval = 1.08-2.00, p = 0.014, respectively) and significance withstands after the Bonferroni correction. No significant age and gender differences between cases and controls were observed. In silico, gene expression analysis showed that the SMAD1 mRNA level was downregulated in the colon and rectal cancer tissues compared to healthy tissues. In conclusion, our findings indicate that SMAD1 rs11100883 and rs7661162 polymorphisms are responsible for increasing the susceptibility of colorectal cancer development in the Bangladeshi population.

TRPM7 Upregulate the Activity of SMAD1 through PLC Signaling Way to Promote Osteogenesis of hBMSCs.

TRPM7 is a member of the transient receptor potential cation channel (TRP channel) subfamily M and possesses both an ion channel domain and a functional serine/threonine α-kinase domain. It has been proven to play an essential role in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). However, the signaling pathway and molecular mechanism for TRPM7 in regulating osteogenic differentiation remain largely unknown. In this study, the potential role and mechanism of TRPM7 in the osteogenic differentiation of hBMSCs were investigated. The results showed that the expression of TRPM7 mRNA and protein increased, as did the osteogenic induction time. Upregulation or inhibition of TRPM7 could promote or inhibit the osteogenic differentiation of hBMSCs for 14 days. It was also found that the upregulation or inhibition of TRPM7 promoted or inhibited the activity of PLC and SMAD1, respectively, during osteogenic differentiation. PLC could promote osteogenic differentiation by upregulating the activity of SMAD1. However, inhibition of PLC alone could reduce the activity of SMAD1 but not inhibit completely the activation of SMAD1. Therefore, we inferred that it is an important signaling pathway for TRPM7 to upregulate the activity of SMAD1 through PLC and thereby promote the osteogenic differentiation of hBMSCs, but it is not a singular pathway. TRPM7 may also regulate the activation of SMAD1 through other ways, except for PLC, during osteogenic differentiation of hBMSCs.

NGAL and SMAD1 gene expression in the early detection of diabetic nephropathy by liquid biopsy.

INTRODUCTION: Diabetic nephropathy (DN) is a disease that progresses with the slow and progressive decline of the glomerular filtration rate (GFR); the installation of this pathology is silent and one of the major causes of death in patients with diabetes. AIMS: To identify new molecular biomarkers for early identification of the onset of DN in patients with type II diabetes mellitus (DM2). We studied the expression profile of the genes; suppressor of mothers against decapentaplegic type 1 (SMAD1), neutrophil gelatinase-associated lipocalin (NGAL) and type IV collagen (COLIV1A) in peripheral blood and urine sediment samples. METHODS: Ninety volunteers, 51 with DM2 and 39 healthy, were recruited from the Faculdade de Medicina do ABC outpatient clinic. We conducted an interview and collected anthropometric data, as well as blood and urine samples for biochemical evaluation and real-time PCR amplification of the genes of interest. RESULTS: Gene expression data: peripheral blood NGAL (DM2 0.09758±0.1914 vs CTL 0.02293±0.04578), SMAD1 (blood: DM2 0.01102±0.04059* vs CTL 0.0001317±0.0003609; urine: DM2 0.7195±2.344* vs CTL 0.09812±0.4755), there was no significant expression of COLIV1A. These genes demonstrated good sensitivity and specificity in the receiving operating characteristic curve evaluation. CONCLUSION: Our data suggest the potential use of NGAL and SMAD1 gene expression in peripheral blood and urine samples as early biomarkers of DN.

Impaired SMAD1/5 Mechanotransduction and Cx37 (Connexin37) Expression Enable Pathological Vessel Enlargement and Shunting.

OBJECTIVE: Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in AVM development, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused AVM-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5, addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions. CONCLUSIONS: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.

Over-expression of miR-187 inhibited cell proliferation and metastasis of glioma via down-regulating SMAD1.

OBJECTIVE: MicroRNAs (miRNAs) have been identified to participate in the tumorigenesis and progression of glioma. However, the expression and function of miR-187 have not been fully elucidated in glioma so far. Therefore, the aim of this study was to investigate the role of miR-187 in glioma and to explore the possible underlying mechanism. PATIENTS AND METHODS: The expression levels of miR-187 in 67 glioma tissues and 21 normal brain tissues, as well as 4 glioma-derived cell lines were measured using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). MiR-187 was overexpressed or inhibited in U251 or U87MG cells using miR-187 mimics or inhibitor transfection, respectively. Colony formation assay and Cell Counting Kit-8 (CCK-8) assay were employed to detect the proliferation ability of cells. Meanwhile, transwell assay and wound-healing assay were applied to evaluate the invasion and migration capacities of cells. Furthermore, Dual-Luciferase assay and Western blot analysis were used to verify the downstream target gene of miR-187 in glioma. RESULTS: MiR-187 expression was significantly lower in glioma tissues and cells when compared with normal brain tissues and cell lines. Up-regulation of miR-187 markedly reduced the proliferation, migration and invasion of U251 cells compared with the negative control group. However, down-regulation of miR-187 remarkably accelerated U87MG cell growth and metastasis compared with inhibitor negative control group. Furthermore, SMAD1 was identified as a direct target for miR-187 in glioma, which could be repressed by miR-187. In addition, over-expression of SMAD1 restored the influence of miR-187 mimics in glioma cells. CONCLUSIONS: MiR-187 was lowly expressed in glioma tissues and cell lines. Acting as a tumor suppressor, miR-187 inhibited cell growth, invasion, and migration in glioma via repressing SMAD1 expression. Our findings might provide a novel insight into the biological diagnosis and treatment in glioma.

Pilose antler aqueous extract promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by stimulating the BMP-2/Smad1, 5/Runx2 signaling pathway.

Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 µg·mL-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.

Pharmacological DNA demethylation restores SMAD1 expression and tumor suppressive signaling in diffuse large B-cell lymphoma.

The sphingosine-1-phosphate (S1P) receptor S1PR2 and its downstream adaptor Gα13 are recurrently mutationally inactivated in the germinal center B-cell subtype of diffuse large B-cell lymphoma (DLBCL) and are silenced by the S1PR2 repressor FOXP1 in the activated B-cell like subtype of the disease. Loss of S1PR2 signaling relieves the germinal center confinement that is maintained by an S1P gradient and allows cells to resist S1P-induced apoptosis. We have shown previously that S1PR2 expression is induced in normal B cells through a newly described transforming growth factor-ß (TGF-ß)/TGF-ßRII/SMAD1 signaling axis that is inactivated in >85% of DLBCL patients. DLBCL cell lines lacking S1PR2, TGFBRII, or SMAD1 as the result of genomic editing all have a strong growth advantage in vitro, as well as in subcutaneous and orthotopic xenotransplantation models. Here, we show that the TGF-ß signaling pathway in DLBCL is blocked at the level of SMAD1 in DLBCL cell lines and patient samples by hypermethylation of CpG-rich regions surrounding the SMAD1 transcription start site. The pharmacologic restoration of SMAD1 expression by the demethylating agent decitabine (DAC) sensitizes cells to TGF-ß-induced apoptosis and reverses the growth of initially SMAD1- cell lines in ectopic and orthotopic models. This effect of DAC is reduced in a SMAD1-knockout cell line. We further show that DAC restores SMAD1 expression and reduces the tumor burden in a novel patient-derived orthotopic xenograft model. The combined data lend further support to the concept of an altered epigenome as a major driver of DLBCL pathogenesis.