Exosomal-miR-522-3p derived from cancer-associated fibroblasts accelerates tumor metastasis and angiogenesis via repression bone morphogenetic protein 5 in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a gastrointestinal tract malignancy. Exosomes secreted by cancer-associated fibroblasts (CAFs) are reported to participate in tumor progression by delivering noncoding RNA or small proteins. However, the function of exosomal miR-522-3p in CRC remains unclear. METHODS: CAFs were derived from tumor tissues, and exosomes were identified by western blot or TEM/NTA and originated from CAFs/NFs. The viability, invasion, and migration of HUVECs and CRC cells was examined using MTT, Transwell, and wound healing assays, respectively. The molecular interactions were validated using dual luciferase reporter assay and RIP. Xenograft and lung metastasis mouse models were generated to assess tumor growth and metastasis. RESULTS: Exosomes extracted from CAFs/NFs showed high expression of CD63, CD81, and TSG101. CAF-derived exosomes significantly increased the viability, angiogenesis, invasion, and migration of HUVECs and CRC cells, thereby aggravating tumor growth, invasion, and angiogenesis in vivo. miR-522-3p was upregulated in CAF-derived exosomes and CRC tissues. Depletion of miR-522-3p reversed the effect of exosomes derived from CAFs in migration, angiogenesis, and invasion of HUVECs and CRC cells. Furthermore, bone morphogenetic protein 5 (BMP5) was identified as a target gene of miR-522-3p, and upregulation of BMP5 reversed the promoting effect of miR-522-3p mimics or CAF-derived exosomes on cell invasion, migration, and angiogenesis of HUVECs and CRC cells. CONCLUSION: Exosomal miR-522-3p from CAFs promoted the growth and metastasis of CRC through downregulating BMP5, which might provide new strategies for the treatment of CRC.

BMP5 ameliorates diabetic peripheral neuropathy by augmenting mitochondrial function and inhibiting apoptosis in Schwann cells.

Diabetic peripheral neuropathy is a common and serious complication of diabetes. Bone morphogenetic protein 5 (BMP5) is a multifunctional protein involved in the nervous system. Nevertheless, its effect on diabetic peripheral neuropathy remained uncharacterized. In this study, diabetic neuropathy in mice was induced by a single dose of 150 mg/kg streptozotocin (STZ) via intraperitoneal injection. Lentivirus expressing BMP5 (LV-BMP5) administration improved pain sensitivity, nerve conduction velocities and morphological alterations of the sciatic nerve of diabetic mice. Elevated BMP5 by LV-BMP5 suppressed cell apoptosis in the sciatic nerve, as evidenced by declined TUNEL-positive cells and down-regulated cleaved caspase-3 and cleaved caspase-9 levels. BMP5 enhanced mitochondrial membrane potential and ATP level. BMP5 also increased the phosphorylation of Smad1/5/9. Besides, the role of BMP5 in high glucose (HG)-stimulated Schwann cells was determined. Results of in vitro studies were in line with the in vivo findings. These experimental data seem to imply that BMP5 prevents the development of diabetic neuropathy via the maintenance of Smad1/5/9-mediated mitochondrial function.

Bmp5 Mutation Alters mRNA Expression During External Ear Development.

To understand changes in gene regulation and mRNA expression in external ear development, we used a bone morphogenetic protein 5 (BMP5) short-ear mouse model. External ear tissues at E15.5 and E17.5 were collected, and mRNA expression profiles were analyzed. Upregulated and downregulated mRNA expression was identified using find_circ and CIRI2 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the differentially expressed mRNAs. Alterations in related signal pathways were identified from the upregulated and downregulated mRNA transcripts. The results showed a correlation between the mRNA expression during external ear development in BMP5 short-ear mice, including key regulatory mRNA changes after point mutations of the Bmp5 gene. This study provides evidence for the mechanism underlying mRNA regulation during external ear development. Changes in mRNA expression profiles also provide clues for future studies regarding the regulatory mechanisms underlying external ear development.

Changes in the Transcriptome-Associated Co-Expression Profile of Embryonic External Ear Development After the BMP5 Gene Mutation.

This study aimed to perform an association analysis of the full transcriptome in Bmp5 short-ear mice during the development of the external ear in mouse embryos using advanced sequencing techniques. To understand the changes in gene regulation and expression of BMP5 gene mutations involved in the external ear embryonic development of mice, external ear tissues of mouse embryos developed to E15.5 and E17.5 were obtained using a BMP5 short-ear mouse model. The association analysis of the full transcriptome mainly involved the analysis of lncRNA and mRNA associations, the analysis of lncRNA and miRNA associations, the analysis of miRNA and mRNA associations, the analysis of circRNA and mRNA associations and circRNA, miRNA, and mRNA associations. The results showed that regulation of the full transcriptome is associated with external ear development in BMP5 short-ear mouse embryos, and some key regulatory changes in full transcriptome after BMP5 gene point mutation are different. This study will provide a new clue to investigate the mechanism underlying the regulation of mouse external ear development by the full transcriptome.

Bmp5 Mutation Alters miRNA Expression During Embryonic External Ear Development.

ABSTRACT: To understand the changes in gene regulation and expression of MicroRNA (miRNA) involved in external mouseear embryonic development after point mutation of the Bmp5 gene, the outer ear tissues of developed E15.5 and E17.5 mouse embryos were obtained using a Bmp5 short ear mouse model, and the changes in miRNA expression profiles were detected. Changes in miRNA expression in the experimental and control groups were identified during Bmp5 short ear mouse embryo development at E15.5 and E17.5. GO and Kyoto Encyclopedia of Genes and Genomes functional annotations were performed on differentially expressed miRNAs. Multiple signal pathways related to miRNA expression were enhanced during the development of E15.5 and E17.5 embryos of Bmp5 short-ear mice. Based on the basic characteristics of miRNAs, this study aimed to determine the differential expression of miRNAs in Bmp5 short-ear mice during the development of external ear embryos using advanced sequencing techniques. The results showed differences in some key regulatory miRNA changes after point mutations in the Bmp5 gene. This study provides new insights into the mechanism by which miRNAs regulate the development of the external mouse ear. Changes in miRNA expression profiles can also provide clues for studying the biological regulatory mechanism of external ear embryonic development.

Whole-Genome Sequencing Identifies Two Novel Rare Mutations in BMP5 and BMP2 in Monozygotic Twins With Microtia.

ABSTRACT: Microtia is a rare congenital anomaly of the ear; it is regulated by both genetic and environmental factors. However, the mechanisms underlying its pathogenesis are unknown. In this study, the genomes of 2-year-old twin sisters with right microtia were sequenced using human genome-wide sequencing, an approach useful for identifying mutations in genes responsible for congenital microtia. The phenotypes of the twin sisters included congenital microtia on the right side, abnormal auricle shape in the right external ear, a peanut shape for the residual ear, and complete atresia of the right external auditory canal. In the twin sisters, we identified a previously unknown mutation in BMP5(exon4:c.833- 4C>G), as well as a new mutation (exon2:c.G332T:p.S111I) in BMP2, both of which were confirmed using polymerase chain reaction-based amplification of the corresponding genome regions, followed by first-generation sequencing. The exon4:c.833-4C>G mutation in human BMP5 may be the main cause of microtia in the twin sisters. A pathogenic mutation in human BMP2 (exon2:c.G332T:p.S111I) may be responsible for the facial deformity in the twin sisters. Thus, our study demonstrates the potential of genome-wide sequencing for identifying novel mutations associated with microtia on the whole-genome scale and extends the mutation spectrum of BMP5. Additionally, our data suggest that BMP2 is another pathogenic gene associated with microtia.

Bmp5 Mutation Alters circRNA Expression During Embryonic External Ear Development.

ABSTRACT: Bmp5 mutation can lead to microtia in mice models; however, its underlying mechanism is unclear. We analyzed circular RNA (circRNA) expression changes and associated gene regulation during embryonic development of the mouse's external ear after a point mutation occurred naturally in the BMP5 gene. The outer ear tissues of BMP5 short-eared mouse model embryos at embryonic day (E) 15.5 and E17.5 were subjected to RNA sequencing. Changes in the circRNA expression profile were detected using find_circ and the CiRi2 software. Differentially expressed circRNAs were annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The circRNA expression profile differed between wild-type and mutant mouse embryos. At E15.5, differentially expressed RNAs were involved in the Hippo signaling pathway, whereas those at E17.5 were associated with stem cell pluripotency. Therefore, circRNA is involved in regulating embryonic external ear development, thus providing a basis for studying the biological aspect of its regulation.

G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer.

Epigenetic abnormalities affect tumor progression, as well as gene expression and function. Among the diverse epigenetic modulators, the histone methyltransferase G9a has been focused on due to its role in accelerating tumorigenesis and metastasis. Although epigenetic dysregulation is closely related to tumor progression, reports regarding the relationship between G9a and its possible downstream factors regulating breast tumor growth are scarce. Therefore, we aimed to verify the role of G9a and its presumable downstream regulators during malignant progression of breast cancer. G9a-depleted MCF7 and T47D breast cancer cells exhibited suppressed motility, including migration and invasion, and an improved response to ionizing radiation. To identify the possible key factors underlying these effects, microarray analysis was performed, and a TGF-ß superfamily member, BMP5, was selected as a prominent target gene. It was found that BMP5 expression was markedly increased by G9a knockdown. Moreover, reduction in the migration/invasion ability of MCF7 and T47D breast cancer cells was induced by BMP5. Interestingly, a G9a-depletion-mediated increase in BMP5 expression induced the phosphorylation of Smad proteins, which are the intracellular signaling mediators of BMP5. Accordingly, we concluded that the observed antitumor effects may be based on the G9a-depletion-mediated increase in BMP5 expression and the consequent facilitation of Smad protein phosphorylation.

Upregulated bone morphogenetic protein 5 enhances proliferation and epithelial-mesenchymal transition process in benign prostatic hyperplasia via BMP/Smad signaling pathway.

BACKGROUND: Benign prostatic hyperplasia (BPH) is one of the most common illnesses in aging men. Recent studies found that bone morphogenetic protein 5 (BMP5) is upregulated in BPH tissues, however, the role of BMP5 in the development of BPH has not been examined. The current study aims to elucidate the potential roles of BMP5 and related signaling pathways in BPH. METHODS: Human prostate cell lines (BPH-1, WPMY-1) and human/rat hyperplastic prostate tissues were utilized. Western blot, quantitative real-time polymerase chain reaction, immunofluorescent staining, and immunohistochemical staining were performed. BMP5-silenced and -overexpressed cell models were generated and then cell cycle progression, apoptosis, and proliferation were determined. The epithelial-mesenchymal transition (EMT) was also quantitated. And rescue experiments by BMP/Smad signaling pathway agonist or antagonist were accomplished. Moreover, BPH-related tissue microarray analysis was performed and associations between clinical parameters and expression of BMP5 were analyzed. RESULTS: Our study demonstrated that BMP5 was upregulated in human and rat hyperplastic tissues and localized both in the epithelial and stromal compartments of the prostate tissues. E-cadherin was downregulated in hyperplastic tissues, while N-cadherin and vimentin were upregulated. Overexpression of BMP5 enhanced cell proliferation and the EMT process via phosphorylation of Smad1/5/8, while knockdown of BMP5 induced cell cycle arrest at G0/G1 phase and blocked the EMT process. Moreover, a BMP/Smad signaling pathway agonist and antagonist reversed the effects of BMP5 silencing and overexpression, respectively. In addition, BMP5 expression positively correlated with prostate volume and total prostate-specific antigen. CONCLUSION: Our novel data suggest that BMP5 modulated cell proliferation and the EMT process through the BMP/Smad signaling pathway which could contribute to the development of BPH. However, further studies are required to determine the exact mechanism. Our study also indicated that BMP/Smad signaling may be rediscovered as a promising new therapeutic target for the treatment of BPH.

BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells.

The embryonic formation of midbrain dopaminergic (mDA) neurons in vivo provides critical guidelines for the in vitro differentiation of mDA neurons from stem cells, which are currently being developed for Parkinson's disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway for in vivo specification of mammalian mDA neurons is virtually unknown. Here, we report that BMP5/7-deficient mice (Bmp5-/-; Bmp7-/-) lack mDA neurons due to reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations in Bmp5-/-; Bmp7-/- mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivating SMAD1 in neural stem cells of mice in vivo (Smad1Nes) hampered the differentiation of progenitor cells into mDA neurons by preventing cell cycle exit, especially of TH+SOX6+ (tyrosine hydroxylase, SRY-box 6) and TH+GIRK2+ (potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. BMP5/7 robustly increased the in vitro differentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to threefold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesis in vivo that balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem-cell-derived mDA neurons in vitro, highlighting the importance of sequential BMP/SMAD inhibition and activation in this process.SIGNIFICANCE STATEMENT We identify bone morphogenetic protein (BMP)/SMAD signaling as a novel essential pathway regulating the development of mammalian midbrain dopaminergic (mDA) neurons in vivo and provide insights into the molecular mechanisms of this process. BMP5/7 regulate MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog) expression to direct mDA neurogenesis. Moreover, the BMP signaling component SMAD1 controls the differentiation of mDA progenitors, particularly to substantia nigra neurons, by directing their cell cycle exit. Importantly, BMP5/7 increase robustly the differentiation of human induced pluripotent and induced neural stem cells to mDA neurons. BMP/SMAD are routinely inhibited in initial stages of stem cell differentiation protocols currently being developed for Parkinson's disease cell replacement therapies. Therefore, our findings on opposing roles of the BMP/SMAD pathway during in vitro mDA neurogenesis might improve these procedures significantly.

Alteration of tumor suppressor BMP5 in sporadic colorectal cancer: a genomic and transcriptomic profiling based study.

BACKGROUND: Although the genetic spectrum of human colorectal cancer (CRC) is mainly characterized by APC, KRAS and TP53 mutations, driver genes in tumor initiation have not been conclusively demonstrated. In this study, we aimed to identify novel markers for CRC. METHODS: We performed exome analysis of sporadic colorectal cancer (sCRC) coding regions to screen loss of function (LoF) mutation genes, and carried out systems-level approaches to confirm top rank gene in this study. RESULTS: We identified loss of BMP5 is an early event in CRC. Deep sequencing identified BMP5 was mutated in 7.7% (8/104) of sCRC samples, with 37.5% truncating mutation frequency. Notably, BMP5 negative expression and its prognostic value is uniquely significant in sCRC but not in other tumor types. Furthermore, BMP5 expression was positively correlated with E-cadherin in CRC patients and its dysregulation play a vital role in epithelial-mesenchymal transition (EMT), thus triggering tumor initiation and development. RNA sequencing identified, independent of BMP/Smads pathway, BMP5 signaled though Jak-Stat pathways to inhibit the activation of oncogene EPSTI1. CONCLUSIONS: Our result support a novel concept that the importance of BMP5 in sCRC. The tumor suppressor role of BMP5 highlights its crucial role in CRC initiation and development.

Bmp5 Regulates Neural Crest Cell Survival and Proliferation via Two Different Signaling Pathways.

Neural crest progenitor cells, which give rise to many ectodermal and mesodermal derivatives, must maintain a delicate balance of apoptosis and proliferation for their final tissue contributions. Here we show that zebrafish bmp5 is expressed in neural crest progenitor cells and that it activates the Smad and Erk signaling pathways to regulate cell survival and proliferation, respectively. Loss-of-function analysis showed that Bmp5 was required for cell survival and this response is mediated by the Smad-Msxb signaling cascade. However, the Bmp5-Smad-Msxb signaling pathway had no effect on cell proliferation. In contrast, Bmp5 was sufficient to induce cell proliferation through the Mek-Erk-Id3 signaling cascade, whereas disruption of this signaling cascade had no effect on cell survival. Taken together, our results demonstrate an important regulatory mechanism for bone morphogenic protein-initiated signal transduction underlying the formation of neural crest progenitors. Stem Cells 2017;35:1003-1014.

The depletion of PinX1 involved in the tumorigenesis of non-small cell lung cancer promotes cell proliferation via p15/cyclin D1 pathway.

BACKGROUND: The telomerase/telomere interacting protein PinX1 has been suggested as a tumor suppressor. However, the clinical and biological significance of PinX1 in human non-small cell lung cancer (NSCLC) is unclear. METHODS: PinX1 gene/expression pattern and its association with NSCLC patient survival were analyzed in cBioportal Web resource and two cohorts of NSCLC samples. A series of in vivo and in vitro assays were performed to elucidate the function of PinX1 on NSCLC cells proliferation and underlying mechanisms. RESULTS: More frequency of gene PinX1 homozygous deletion and heterozygote deficiency was first retrieved from cBioportal Web resource. Low expression of PinX1 correlated with smoking condition, histological type, T stage, N stage, M stage and TNM stage, and was an independent predictor for overall survival in a learning cohort (n = 93) and a validation cohort (n = 51) of NSCLC patients. Furthermore, knockdown of PinX1 dramatically accelerated NSCLC cell proliferation and G1/S transition, whereas ectopic overexpression of PinX1 substantially inhibited cell viability and cell cycle transition in vitro and in vivo. p15/cyclin D1 pathway and BMP5 might contribute to PinX1-associated cell proliferation and cell cycle transition. CONCLUSION: The cost-effective expression of PinX1 could constitute a novel molecular predictor/marker for NSCLC management.

Evaluation of the association between a single-nucleotide polymorphism of bone morphogenetic proteins 5 gene and risk of knee osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a chronic degenerative disorder probably affected by both genetic and environmental causes. Bone morphogenetic proteins (BMPs) are bone-derived factors that can induce new bone formation. Single-nucleotide polymorphisms (SNPs) of BMP5 gene alters the transcriptional activity of the BMP5 promoter that has been involved in OA susceptibility. This case-control study investigated the association of rs1470527 and rs9382564 SNP of BMP5 gene with susceptibility to knee OA (KOA). MATERIALS AND METHODS: A total of 499 cases with radiographic KOA and 458 age- and sex-matched healthy controls were enrolled. Venous blood samples were obtained from all the cases as well as controls for polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The genotype distribution for rs1470527 and rs9382564 SNP was significantly different in cases and controls (P < 0.0001). Within both the SNPs of BMP5 gene, genotype CT and TT were significantly (P < 0.0001) associated with KOA as compared to the CC genotype. T allele of both the studied SNP was significantly associated with KOA (P < 0.0001). The allele frequencies of rs1470527 were 0.56(T) and 0.44(C) in cases and 0.33(T) and 0.67(C) in controls and in rs9382564 were 0.57(C) and 0.43(T) in cases and 0.71(C) and 0.29(T) in controls. Further in relation with clinical severity of OA, we observed signification association of TT genotype with both visual analog scale (P < 0.0001) and Western Ontario and McMaster Universities score (P < 0.05). CONCLUSION: Our results indicate significant association of rs1470527 and rs9382564 polymorphism of BMP5 gene with KOA.

Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria.

The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized. Changes in extracellular Ca(2+) concentration ([Ca(2+) ]e ), as well as modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPγ-S and high [Ca(2+) ]e (5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPγ-S stimulates cell transition from the G0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 µM ATPγ-S and [Ca(2+) ]e (ATP-[Ca(2+) ]e ) for 48 h increases cell number significantly above the control. ATPγ-S treatment in osteogenic medium containing [Ca(2+) ]e stimulates the gene expression of BMP-4, BMP-5, and OPN at 16, 48, and 72 h, respectively, above control. In same conditions, treatment for 6 days with 10 µM UTP or 100 µM UDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statistically significant increase in calcium deposits at 15 and 18 days, for 10 µM ATPγ-S treatment, and at 18 and 22 days, for [Ca(2+) ]e treatment, respect to control but ATP-[Ca(2+) ]e treatment shown a significant greater mineralization at 15 days respect to ATPγ-S, and at 18 days respect to both agonists. In conclusion, we demonstrated that an osteogenic medium containing 10 µM ATPγ-S and 5.35 mM [Ca(2+) ]e enhance osteogenesis and mineralization by rat primary calvarial cells cultures. J. Cell. Biochem. 117: 2658-2668, 2016. © 2016 Wiley Periodicals, Inc.

Differential expression of bone morphogenetic protein 5 in human lung squamous cell carcinoma and adenocarcinoma.

Bone morphogenetic proteins (BMPs) play important roles in tumor cell proliferation, metastasis, and invasion. However, the expression patterns of BMPs in patients with non-small-cell lung cancer (NSCLC) and their correlations with NSCLC pathogenesis have not been examined yet. In this study, the mRNA levels of BMP family members in NSCLC tissues were analyzed and results showed that the mRNA levels of BMP5 and BMP7 were significantly down-regulated and up-regulated, respectively, in tumor tissues compared with those in the corresponding noncancerous tissues. Interestingly, the mRNA level of BMP5 was significantly higher in lung adenocarcinoma tissues than that in lung squamous cell carcinoma tissues. Furthermore, results from immunohistochemistry analysis confirmed stronger expression of BMP5 protein in lung adenocarcinoma than in lung squamous cell carcinoma. Our findings suggested that BMP5 might be a potential prognostic biomarker or therapeutic target for patients with NSCLC.

eEOC-mediated modulation of endothelial autophagy, senescence, and EnMT in murine diabetic nephropathy.

Diabetic nephropathy is the most frequent single cause of end-stage renal disease in our society. Microvascular damage is a key event in diabetes-associated organ malfunction. Early endothelial outgrowth cells (eEOCs) act protective in murine acute kidney injury. The aim of the present study was to analyze consequences of eEOC treatment of murine diabetic nephropathy with special attention on endothelial-to-mesenchymal transdifferentiation, autophagy, senescence, and apoptosis. Male C57/Bl6N mice (8-12 wk old) were treated with streptozotocin for 5 consecutive days. Animals were injected with untreated or bone morphogenetic protein (BMP)-5-pretreated syngeneic murine eEOCs on days 2 and 5 after the last streptozotocin administration. Four, eight, and twelve weeks later, animals were analyzed for renal function, proteinuria, interstitial fibrosis, endothelial-to-mesenchymal transition, endothelial autophagy, and senescence. In addition, cultured mature murine endothelial cells were investigated for autophagy, senescence, and apoptosis in the presence of glycated collagen. Diabetes-associated renal dysfunction (4 and 8 wk) and proteinuria (8 wk) were partly preserved by systemic cell treatment. At 8 wk, antiproteinuric effects were even more pronounced after the injection of BMP-5-pretreated cells. The latter also decreased mesenchymal transdifferentiation of the endothelium. At 8 wk, intrarenal endothelial autophagy (BMP-5-treated cells) and senescence (native and BMP-5-treated cells) were reduced. Autophagy and senescence in/of cultured mature endothelial cells were dramatically reduced by eEOC supernatant (native and BMP-5). Endothelial apoptosis decreased after incubation with eEOC medium (native and BMP-5). eEOCs act protective in diabetic nephropathy, and such effects are significantly stimulated by BMP-5. The cells modulate endothelial senescence, autophagy, and apoptosis in a protective manner. Thus, the renal endothelium could serve as a therapeutic target in diabetes-associated kidney dysfunction.

ATP and UTP stimulate bone morphogenetic protein-2,-4 and -5 gene expression and mineralization by rat primary osteoblasts involving PI3K/AKT pathway.

The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast. On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation. In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100µM ATP, ATPγS and UTP, but not ADP, ADPßS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100µM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Analysis of human alveolar osteoblast behavior on a nano-hydroxyapatite substrate: an in vitro study.

BACKGROUND: Nano-hydroxyapatite (nHA) is a potential ideal biomaterial for bone regeneration. However, studies have yet to characterize the behavior of human osteoblasts derived from alveolar bone on nHA. Thus, the aim of the present study was to evaluate the influence of nHA on the adhesion, proliferation and differentiation of these alveolar bone-derived cells. METHODS: Primary human alveolar osteoblasts were collected from the alveolar ridge of a male periodontal patient during osseous resective surgery and grown on culture plates coated with either polylysine or polylysine with nano-hydroxyapatite (POL/nHA) composite. The cells were grown and observed for 14 days, and then assessed for potential modifications to osteoblasts homeostasis as evaluated by quantitative reverse transcriptase-polymerase chain reaction (real time RT-PCR), scanning electron microscopy and atomic force microscopy. RESULTS: Real time PCR revealed a significant increase in the expression of the selected markers of osteoblast differentiation (bone morphogenetic protein (BMP)-2,-5,-7, ALP, COLL-1A2, OC, ON) in cells grown on the POL/nHA substrate. In addition, as compared with the POL surface, cells grown on the POL/nHA substrate demonstrated better osteoconductive properties, as demonstrated by the increase in adhesion and spreading, likely as a result of the increased surface roughness of the composite. CONCLUSIONS: The increased expression of BMPs and osteoinductive biomarkers suggest that nano-hydroxyapatite may stimulate the proliferation and differentiation of local alveolar osteoblasts and thus encourage bone regeneration at sites of alveolar bone regeneration.

Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state.

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFß), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)-a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.