Search Results (52)

Berberine has been widely studied for its biological functions in various diseases, including cancer, diabetes, and cardiovascular diseases. Nevertheless, structural modifications of berberine have been demonstrated to augment its pharmacological efficacy in specific biological processes, particularly osteogenesis. In this study, we aimed to explore new berberine derivatives with pro-osteogenic activity and molecular mechanisms. Our results demonstrated that compound 13 is the most effective among the tested compounds. Compound 13 significantly enhanced BMP4-induced alkaline phosphatase (ALP) staining and increased the transcriptional activity of osteogenic markers such as ALP, Runt-related gene 2 (Runx2), and Osterix at both the mRNA and protein levels. Furthermore, we found that the Akt and PKC signaling pathways play crucial roles in compound 13-induced osteogenesis via treatment with specific inhibitors. The molecular docking results supported the potential interaction between compound 13 and these kinases. These findings highlighted the regulatory role of compound 13 in osteoblast differentiation via the Akt and PKC signaling pathways. Overall, our study provides compelling evidence that compound 13 is a promising therapeutic candidate for the treatment of osteoporosis, with the potential for further development and optimization to improve bone health and strength. Full article

The metabolic poise, or balance, between glycolysis and fatty acid oxidation (FAO) has recently been found to play a critical role in osteogenic differentiation and homeostasis. While simulated microgravity (SMG) is known to impede osteoblast differentiation (OBD) by inhibiting the Wnt/β-catenin pathway, how it affects osteoblast metabolism in this context remains unclear. We previously analyzed the effect of SMG on the differentiation of pre-osteoblast MC3T3-E1 cells and found that it reduced focal adhesion kinase (FAK) activity. This, in turn, downregulated Wnt/β-catenin and two of its downstream targets critical for OBD bone morphogenic protein-2 (BMP2) and type-1 collagen (COL1) formation, leading to a reduction in alkaline phosphatase (ALP) activity and cell matrix mineralization. In this study, we further analyzed how SMG-induced alterations in energy metabolism contribute to the inhibition of OBD in MC3T3-E1 cells. Consistent with our earlier findings, we demonstrated that SMG inhibits OBD by downregulating the collective activity of FAK and the Wnt/β-catenin-BMP2-COL1 transcriptional pathway. Interestingly, we observed that SMG also reduces the abundance of sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and carnitine palmitoyl transferase-1α (CPT1A), which are all key metabolic factors regulating mitochondrial number and FAO capacity. Accordingly, we found that the mitochondrial content and FAO potential of MC3T3-E1 cells were lower upon exposure to SMG but were both rescued upon administration of the FAK activator cytotoxic necrotizing factor-1 (CNF1), thereby allowing cells to overcome SMG-induced inhibition of OBD. Taken together, our study indicates that the metabolic regulator SIRT1 may be a new target for reversing SMG-induced bone loss. Full article

Endothelial cells (ECs) respond to concurrent stimulation by biochemical factors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemorrhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. However, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS microfluidic device and a custom image analysis pipeline. We extended the previously reported synergy between SS and BMP9 to include BMP10 and BMP9/10. Moreover, we demonstrated that this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm²) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1’s basal activity and response to minimal ligand levels depend on endocytosis, distinct from cell–cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. However, an in-depth analysis of ALK1 receptor trafficking’s molecular mechanisms requires further investigation. Full article

Bone fracture healing is a complex biological process involving four phases coordinated over time: hematoma formation, granulation tissue formation, bony callus formation, and bone remodelling. Bone fractures represent a significant health problem, particularly among the elderly population and patients with comorbidities. Therapeutic strategies proposed to treat such fractures include the use of autografts, allografts, and tissue engineering strategies. It has been shown that bone morphogenetic protein 2 (BMP-2) has a therapeutic potential to enhance fracture healing. Despite the clinical efficacy of BMP-2 in osteoinduction and bone repair, adverse side effects and complications have been reported. Therefore, in this in vitro study, we propose the use of a disaccharide compound (DP2) to improve the mineralisation process. We first evaluated the effect of DP2 on primary human osteoblasts (HOb), and then investigated the mechanisms involved. Our findings showed that (i) DP2 improved osteoblast differentiation by inducing alkaline phosphatase activity, osteopontin, and osteocalcin expression; (ii) DP2 induced earlier in vitro mineralisation in HOb cells compared to BMP-2 mainly by earlier activation of Runx2; and (iii) DP2 is internalized in HOb cells and activates the protein kinase C signalling pathway. Consequently, DP2 is a potential therapeutical candidate molecule for bone fracture repair. Full article

When research on osteogenic differentiation in dental follicle cells (DFCs) began, projects focused on bone morphogenetic protein (BMP) signaling. The BMP pathway induces the transcription factor DLX3, whichh in turn induces the BMP signaling pathway via a positive feedback mechanism. However, this BMP2/DLX3 signaling pathway only seems to support the early phase of osteogenic differentiation, since simultaneous induction of BMP2 or DLX3 does not further promote differentiation. Recent data showed that inhibition of classical protein kinase C (PKCs) supports the mineralization of DFCs and that osteogenic differentiation is sensitive to changes in signaling pathways, such as protein kinase B (PKB), also known as AKT. Small changes in the lipidome seem to confirm the participation of AKT and PKC in osteogenic differentiation. In addition, metabolic processes, such as fatty acid biosynthesis, oxidative phosphorylation, or glycolysis, are essential for the osteogenic differentiation of DFCs. This review article attempts not only to bring the various factors into a coherent picture of osteogenic differentiation in DFCs, but also to relate them to recent developments in other types of osteogenic progenitor cells. Full article

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrosis and, thus, build the “soil” for hepatocarcinogenesis. Furthermore, HSCs are known to promote the progression of hepatocellular carcinoma (HCC), but the molecular mechanisms are only incompletely understood. Recently, we newly described the expression of bone morphogenetic protein 13 (BMP13) by HSCs in fibrotic liver tissue. In addition, BMP13 has mostly been studied in the context of cartilage and bone repair, but not in liver disease or cancer. Thus, we aimed to analyze the expression and function of BMP13 in HCC. Expression analyses revealed high BMP13-expression in activated human HSCs, but not in human HCC-cell-lines. Furthermore, analysis of human HCC tissues showed a significant correlation between BMP13 and α-smooth muscle actin (α-SMA), and immunofluorescence staining confirmed the co-localization of BMP13 and α-SMA, indicating activated HSCs as the cellular source of BMP13 in HCC. Stimulation of HCC cells with recombinant BMP13 increased the expression of the inhibitors of differentiation 1 (ID1) and 2 (ID2), which are known targets of BMP-signaling and cell-cycle promotors. In line with this, BMP13-stimulation caused an induced SMAD 1/5/9 and extracellular signal-regulated kinase (ERK) phosphorylation, as well as reduced expression of cyclin-dependent kinase inhibitors 1A (CDKN1A) and 2A (CDKN2A). Furthermore, stimulation with recombinant BMP13 led to increased proliferation and colony size formation of HCC cells in clonogenicity assays. The protumorigenic effects of BMP13 on HCC cells were almost completely abrogated by the small molecule dorsomorphin 1 (DMH1), which selectively blocks the intracellular kinase domain of ALK2 and ALK3, indicating that BMP13 acts via these BMP type I receptors on HCC cells. In summary, this study newly identifies stroma-derived BMP13 as a potential new tumor promotor in HCC and indicates this secreted growth-factor as a possible novel therapeutic target in HCC. Full article

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis. Full article

The genetic makeup of the triple-wild-type melanoma (BRAF, NRAS and NF1) has been known for some time, but those studies grouped together rare histopathological versions with common ones, as well as mucosal and even uveal ones. Here we used whole genome sequencing to genetically characterize the triple-wild-type melanoma (TWM), termed here as BRAF, RAS and KIT wild type (the most frequent oncogenic drivers of skin melanoma), using the most common histological forms and excluding rare ones. All these tumors except one were clearly induced by UV based on the mutational signature. The tumor mutational burden was low in TWM, except in the NF1 mutant forms, and a relatively high frequency of elevated LOH scores suggested frequent homologue recombination deficiency, but this was only confirmed by the mutation signature in one case. Furthermore, all these TWMs were microsatellite-stabile. In this driverless setting, we revealed rare oncogenic drivers known from melanoma or other cancer types and identified rare actionable tyrosine kinase mutations in NTRK1, RET and VEGFR1. Mutations of TWM identified genes involved in antitumor immunity (negative and positive predictors of immunotherapy), Ca⁺⁺ and BMP signaling. The two regressed melanomas of this cohort shared a 17-gene mutation signature, containing genes involved in antitumor immunity and several cell surface receptors. Even with this comprehensive genomic approach, a few cases remained driverless, suggesting that unrecognized drivers are hiding among passenger mutations. Full article

The Drosophila lymph gland is an ideal model for studying hematopoiesis, and unraveling the mechanisms of Drosophila hematopoiesis can improve our understanding of the pathogenesis of human hematopoietic malignancies. Bone morphogenetic protein (BMP) signaling is involved in a variety of biological processes and is highly conserved between Drosophila and mammals. Decapentaplegic (Dpp)/BMP signaling is known to limit posterior signaling center (PSC) cell proliferation by repressing the protooncogene dmyc. However, the role of two other TGF-β family ligands, Glass bottom boat (Gbb) and Screw (Scw), in Drosophila hematopoiesis is currently largely unknown. Here, we showed that the loss of Gbb in the cortical zone (CZ) induced lamellocyte differentiation by overactivation of the EGFR and JNK pathways and caused excessive differentiation of plasmatocytes, mainly by the hyperactivation of EGFR. Furthermore, we found that Gbb was also required for preventing the hyperproliferation of the lymph glands by inhibiting the overactivation of the Epidermal Growth Factor Receptor (EGFR) and c-Jun N-terminal Kinase (JNK) pathways. These results further advance our understanding of the roles of Gbb protein and the BMP signaling in Drosophila hematopoiesis and the regulatory relationship between the BMP, EGFR, and JNK pathways in the proliferation and differentiation of lymph gland hemocytes. Full article

A prospective source of stem cells for bone tissue engineering is adipose-derived stem cells (ADSCs), and BMP-2 has been proven to be highly effective in promoting the osteogenic differentiation of stem cells. Rarely has research been conducted on the impact of lactoferrin (LF) on ADSCs’ osteogenic differentiation. As such, in this study, we examined the effects of LF and BMP-2 to assess the ability of LF to stimulate ADSCs’ osteogenic differentiation. The osteogenic medium was supplemented with the LF at the following concentrations to culture ADSCs: 0, 10, 20, 50, 100, and 500 μg/mL. The Cell Counting Kit-8 (CCK-8) assay was used to measure the proliferation of ADSCs. Calcium deposition, alkaline phosphatase (ALP) staining, real-time polymerase chain reaction (RT-PCR), and an ALP activity assay were used to establish osteogenic differentiation. RNA sequencing analysis was carried out to investigate the mechanism of LF boosting the osteogenic development of ADSCs. In the concentration range of 0–100 μg/mL, LF concentration-dependently increased the proliferative vitality and osteogenic differentiation of ADSCs. At a dose of 500 μg/mL, LF sped up and enhanced differentiation, but inhibited ADSCs from proliferating. LF (100 and 500 μg/mL) produced more substantial osteoinductive effects than BMP-2. The PI3 kinase/AKT (PI3K/AKT) and IGF-R1 signaling pathways were significantly activated in LF-treated ADSCs. The in vitro study results showed that LF could effectively promote osteogenic differentiation of ADSCs by activating the PI3K/AKT and IGF-R1 pathways. In our in vitro investigation, an LF concentration of 100 μg/mL was optimal for osteoinduction and proliferation. Our study suggests that LF is an attractive alternative to BMP-2 in bone tissue engineering. As a bioactive molecule capable of inducing adipose stem cells to form osteoblasts, LF is expected to be clinically used in combination with biomaterials as an innovative molecular and cellular therapy to promote bone repair. Full article

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not—e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased β3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine. Full article

Differentiating mesenchymal stromal cells (MSCs) into articular chondrocytes (ACs) for application in clinical cartilage regeneration requires a profound understanding of signaling pathways regulating stem cell chondrogenesis and hypertrophic degeneration. Classifying endochondral signals into drivers of chondrogenic speed versus hypertrophy, we here focused on insulin/insulin-like growth factor 1 (IGF1)-induced phosphoinositide 3-kinase (PI3K)/AKT signaling. Aware of its proliferative function during early but not late MSC chondrogenesis, we aimed to unravel the late pro-chondrogenic versus pro-hypertrophic PI3K/AKT role. PI3K/AKT activity in human MSC and AC chondrogenic 3D cultures was assessed via Western blot detection of phosphorylated AKT. The effects of PI3K inhibition with LY294002 on chondrogenesis and hypertrophy were assessed via histology, qPCR, the quantification of proteoglycans, and alkaline phosphatase activity. Being repressed by ACs, PI3K/AKT activity transiently rose in differentiating MSCs independent of TGFβ or endogenous BMP/WNT activity and climaxed around day 21. PI3K/AKT inhibition from day 21 on equally reduced chondrocyte and hypertrophy markers. Proving important for TGFβ-induced SMAD2 phosphorylation and SOX9 accumulation, PI3K/AKT activity was here identified as a required stage-dependent driver of chondrogenic speed but not of hypertrophy. Thus, future attempts to improve MSC chondrogenesis will depend on the adequate stimulation and upregulation of PI3K/AKT activity to generate high-quality cartilage from human MSCs. Full article

Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We found that TB-CM promoted the osteogenic differentiation of MSCs in a dose-dependent manner. Furthermore, it inhibited adipogenesis of MSCs. We also found that the primary trophoblast-derived conditioned medium (PTB-CM) significantly enhanced the proliferation and osteogenic differentiation of human MSCs. Our study demonstrated the regulatory mechanisms underlying the TB-CM-induced osteogenesis in MSCs. An upregulation of genes associated with cytokines/chemokines was observed. The treatment of MSCs with TB-CM stimulated osteogenesis by activating several biological processes, such as mitogen-activated protein kinase (MAPK) and bone morphogenetic protein 2 (BMP2) signaling. This study demonstrated the proliferative and osteogenic efficacies of the trophoblast-derived secretomes, suggesting their potential for use in clinical interventions for bone regeneration and treatment. Full article

Bone morphogenetic proteins (BMPs) are growth factors that have a vital role in the production of bone, cartilage, ligaments, and tendons. Tumors’ upregulation of bone morphogenetic proteins (BMPs) and their receptors are key features of cancer progression. Regulation of the BMP kinase system is a new promising strategy for the development of anti-cancer drugs. In this work, based on a careful literature study, a library of benzothiophene and benzofuran derivatives was subjected to different computational techniques to study the effect of chemical structure changes on the ability of these two scaffolds to target BMP-2 inducible kinase, and to reach promising candidates with proposed activity against BMP-2 inducible kinase. The results of screening against Lipinski’s and Veber’s Rules produced twenty-one outside eighty-four compounds having drug-like molecular nature. Computational ADMET studies favored ten compounds (11, 26, 27, 29, 30, 31, 34, 35, 65, and 72) with good pharmacokinetic profile. Computational toxicity studies excluded compound 34 to elect nine compounds for molecular docking studies which displayed eight compounds (26, 27, 29, 30, 31, 35, 65, and 72) as promising BMP-2 inducible kinase inhibitors. The nine fascinating compounds will be subjected to extensive screening against serine/threonine kinases to explore their potential against these critical proteins. These promising candidates based on benzothiophene and benzofuran scaffolds deserve further clinical investigation as BMP-2 kinase inhibitors for the treatment of cancer. Full article

Degenerative disc disease (DDD) is an important cause of low back pain. Repetitive tensile stress from the daily motion of the spine predisposes it to injury of the annulus fibrosus (AF) which causes IVD degeneration. This study aims to determine the causal relationship between mechanical stretch and osteogenesis in the AF cells of IVD as affected by bone morphogenic proteins (BMPs), specifically BMP-2/6 heterodimers. Our results found that 15% tensile stress (high cyclic stretching, HCS) may induce the expression of osteogenesis-related markers (Runx2, osterix) by upregulating BMP-2/6 heterodimeric ligands and their receptors on the human AF cell line. HCS also induced transient phosphorylation of p38 mitogen-activated protein (MAP) kinase and SMAD1/5/8. Neutralizing antibodies to the BMP-2/6 receptor (ALK3) blocked the expression of Runx2 and osterix, as well as the phosphorylation of p38 and SMAD1/5/8. In addition, treatment with a p38 MAPK inhibitor (SB203580) or siRNA to neutralize the effects of SMAD1/5/8 suppressed tensile stress-induced Runx2 and osterix expression. Mechanical stretching induces activation of p38 MAP kinase and SMAD1/5/8 signaling pathways, followed by the upregulation of BMP-2/6 heterodimer expression, thereby stimulating osteogenic Runx2 and osterix expression on AF cells. HCS may accelerate the progression of IVD degeneration by promoting an osteogenic response. Full article