Regulation of myogenic progenitor proliferation in human fetal skeletal muscle by BMP4 and its antagonist Gremlin

Skeletal muscle side population (SP) cells are thought to be “stem”-like cells. Despite reports confirming the ability of muscle SP cells to give rise to differentiated progeny in vitro and in vivo, the molecular mechanisms defining their phenotype remain unclear. In this study, gene expression analyses of human fetal skeletal muscle demonstrate that bone morphogenetic protein 4 (BMP4) is highly expressed in SP cells but not in main population (MP) mononuclear muscle-derived cells. Functional studies revealed that BMP4 specifically induces proliferation of BMP receptor 1a–positive MP cells but has no effect on SP cells, which are BMPR1a-negative. In contrast, the BMP4 antagonist Gremlin, specifically up-regulated in MP cells, counteracts the stimulatory effects of BMP4 and inhibits proliferation of BMPR1a-positive muscle cells. In vivo, BMP4-positive cells can be found in the proximity of BMPR1a-positive cells in the interstitial spaces between myofibers. Gremlin is expressed by mature myofibers and interstitial cells, which are separate from BMP4-expressing cells. Together, these studies propose that BMP4 and Gremlin, which are highly expressed by human fetal skeletal muscle SP and MP cells, respectively, are regulators of myogenic progenitor proliferation.

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CSIG-30. miR-146a INHIBITS TUMORIGENESIS AND INDUCES TEMOZOLOMIDE SENSITIVITY VIA AFFECTING CANCER STEM CELL PROPERTIES IN GBM

Neuro-Oncology ◽

10.1093/neuonc/noz175.200 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi50-vi50

Author(s):

Tiantian Cui ◽

Erica Hlavin Bell ◽

Joseph McElroy ◽

Kevin Liu ◽

Pooja Manchanda Gulati ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cancer Stem Cell ◽

Molecular Mechanisms ◽

Functional Studies ◽

Average Survival ◽

Novel Biomarkers ◽

The Ohio State University

Abstract BACKGROUND Glioblastomas (GBMs) are the most aggressive primary brain tumors, with an average survival time of less than 15 months. miRNAs are emerging as promising and novel biomarkers in GBM. The aims of this study are: 1) to investigate novel miRNAs biomarkers that affect tumorigenesis and therapeutic sensitivity, and 2) to study the underlying molecular mechanisms in GBM. METHODS Nanostring v3 was performed followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a in GBM tumorigenesis and therapeutic response and the molecular mechanisms were investigated. RESULTS UVA analyses demonstrated that miR-146a is one of the top miRNAs that correlated with better prognosis in GBM patients (p=9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p< 0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p=0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p< 0.01) and prolonged survival (p=0.001) in vivo. In addition, miR-146a is downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly affected glioma cancer stem cell self-renewal. We also found that overexpression of miR-146a significantly inhibited the NF-κB, AKT, and ERK pathways. CONCLUSION Our data suggest, for the first time, that miR-146a predicts favorable prognosis for GBM patients and sensitizes primary GBM cells to TMZ treatment in vitro and in vivo through regulating glioma stem cells. Importantly, miR-146a may prove to be a master switch shutting off AKT, NF-κB, as well as other pathways and may overcome redundancies among these pathways leading to resistance. FUNDING: Bohnenn Fund (to PR), R01CA108633, R01CA169368, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC).

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Endogenous Galectin-3 is Required for Skeletal Muscle Repair

10.1101/2020.10.01.322461 ◽

2020 ◽

Author(s):

Daniel Giuliano Cerri ◽

Lilian Cataldi Rodrigues ◽

Vani Maria Alves ◽

Juliano Machado ◽

Víctor Alexandre Félix Bastos ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Culture ◽

Molecular Mechanisms ◽

Repair Process ◽

Muscle Repair ◽

Galectin 3 ◽

Self Repair ◽

Myoblast Cell

ABSTRACTSkeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian β-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: i) muscle repair in vivo using a chloride-barium myolesion mouse model, and ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 days of the myolesion, while MyoD and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Pax7 detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. These findings suggest that the detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast cell culture compared to Wild Type (WT) cells. We observed Gal-3 expression in WT states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

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Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

10.20944/preprints201904.0277.v1 ◽

2019 ◽

Author(s):

Takehito Sugasawa ◽

Tome Yoshiya ◽

Yoshinori Takeuchi ◽

Naoya Yahagi ◽

Rahul Sharma ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Binding Protein ◽

Cold Stimulation ◽

Vitro Assay ◽

Mitochondrial Dna Copy Number ◽

Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injury. However, its molecular mechanisms are unknown. To clarify these mechanisms, in this study, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cAMP response element-binding protein 1 (CREB1) was markedly phosphorylated (as pCREB1) and CREB-binding protein (CBP) was recruited to pCREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes such as Pgc-1α involved in mitochondrial biogenesis. The foregoing results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes in a manner dependent on cold stimulation frequency and duration. This information may serve as an impetus for further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

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EGR1 Modulated LncRNA HNF1A-AS1 Drives Glioblastoma Progression Via miR-22-3p/ENO1 Axis

10.21203/rs.3.rs-477104/v1 ◽

2021 ◽

Author(s):

Chunchun Ma ◽

Hongliang Wang ◽

Gang Zong ◽

Jie He ◽

Yuyang Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Molecular Mechanisms ◽

Bioinformatic Analysis ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Clinical Samples ◽

Regulation Mechanism ◽

Functional Studies

Abstract Background: Accumulating evidences revealed that long noncoding RNAs (lncRNAs) have been participated in cancer malignant progression, including glioblastoma multiforme (GBM). Despite much studies have found the precise biological role in the regulatory mechanisms of GBM，however the molecular mechanisms，particularly upstream mechanisms still need further elucidated. Methods: RT-QPCR, cell transfection, western blotting and bioinformatic analysis were executed to detect the expression of EGR1, HNF1A-AS1, miR-22-3p and ENO1 in GBM. Cell proliferation assay, colony formation assay, wound healing, migration and invasion assays were performed to detect the malignant characters of GBM cell. The molecular regulation mechanism was confirmed by luciferase reporter assay, ChIP and RIP. Finally, orthotopic mouse models were established to examine the effect of HNF1A-AS1 in vivo.Results: In the current study, we analyzed clinical samples to show that the long non-coding antisense transcript of HNF1A, HNF1A-AS1, is upregulated and associated with poor prognosis in GBM. Functional studies revealed that knockdown of HNF1A-AS1 markedly inhibits cell proliferation, migration and invasion both in vitro and in vivo, whereas overexpression of HNF1A-AS1 exerts opposite effect. Mechanistically, the transcription factor EGR1 forced the transcription of HNF1A-AS1 by directly binding the promoter region of HNF1A-AS1. Furthermore, combined bioinformatics analysis with our mechanistic work, using luciferase reporter assays and RIP, we first demonstrated that HNF1A-AS1 functions as a competing endogenous RNA (ceRNA) with miR-22-3p to regulate ENO1 expression in GBM cells. HNF1A-AS1 directly binds to miR-22-3p and significantly inhibits miR-22-3p expression, while ENO1 expression was increased. miR-22-3p inhibitor offsets the HNF1A-AS1 silencing induced suppression in proliferation, migration and invasion of GBM cells, as well as promotion effect on ENO1 expression. ENO1 was verified as a direct target of miR-22-3p and its expression levels was negatively with the prognosis in GBM patients. Conclusion: Taken together, our study illuminated the definite mechanism of HNF1A-AS1 in promoting GBM malignancy, and provided a novel therapeutic target for further clinical application.

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In Vitro Models to Study Mechanisms of Lung Toxicity

Alternatives to Laboratory Animals ◽

10.1177/026119299001800127.1 ◽

1990 ◽

Vol 18 (1_part_1) ◽

pp. 267-281

Author(s):

Åke Ryrfeldt ◽

Ian A. Cotgreave ◽

Peter Moldéus

Keyword(s):

Molecular Mechanisms ◽

In Vitro Models ◽

Lung Toxicity ◽

Lung Mechanics ◽

Lung Cells ◽

Suitable Model ◽

Clara Cells ◽

Functional Studies

Several functioning in vitro systems of varying complexity are currently in use for the study of mechanisms of lung toxicity. The isolated perfused lung is the model closest to the in vivo situation. It is a suitable model for combining metabolic and functional studies. It is, for instance, possible to relate changes in lung mechanics and lung perfusion flow to the release of various mediators during exposure of the lung to various agents. A simpler model may be constructed from lung slices which are less viable but suitable for uptake as well as metabolism studies. Specific lung cells such as Clara cells and type II pneumocytes have been isolated and cultured and are valuable tools for studies of the molecular mechanisms of lung toxicity, particularly in cases of cell-specific toxicity. There is, however, a great need to develop techniques for the isolation and culture of other types of lung cells and also to improve the culturing techniques for those already isolated.

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Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

International Journal of Molecular Sciences ◽

10.3390/ijms21134588 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4588

Author(s):

Takehito Sugasawa ◽

Yoshiya Tome ◽

Yoshinori Takeuchi ◽

Yasuko Yoshida ◽

Naoya Yahagi ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Binding Protein ◽

Cold Stimulation ◽

Vitro Assay ◽

Mitochondrial Dna Copy Number ◽

Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

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Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01936-4 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Meng Cao ◽

Yi Wang ◽

Yijing Xiao ◽

Dandan Zheng ◽

Chunchun Zhi ◽

...

Keyword(s):

Colorectal Cancer ◽

Clock Gene ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Clock Genes ◽

Tumor Development ◽

Creb Binding Protein ◽

Functional Studies

Abstract Background Colorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated. Methods Immunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis. Results We found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β-catenin signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless. Conclusion Timeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

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FSH secretion predominates in vivo and in vitro in patients with non-functioning pituitary adenomas

Acta Endocrinologica ◽

10.1530/eje.1.01854 ◽

2005 ◽

Vol 152 (3) ◽

pp. 363-370 ◽

Cited By ~ 8

Author(s):

P L Hanson ◽

S J B Aylwin ◽

J P Monson ◽

J M Burrin

Keyword(s):

Cell Cultures ◽

Pituitary Adenomas ◽

Molecular Mechanisms ◽

Pituitary Tumour ◽

Pituitary Cell ◽

Pituitary Cells ◽

Functional Studies ◽

Non Functioning Pituitary Adenomas

Objective: Non-functioning pituitary adenomas (NFPAs) are characterised by the lack of symptoms of hormone hypersecretory syndromes but in vitro studies have demonstrated that tumour cells may stain for gonadotrophins and/or their α- or β-subunits. In this study, we aimed to examine the pattern of secretion of LH and FSH from a series of pituitary adenomas cultured in vitro and where data were available to relate the results to pre-operative serum gonadotrophin levels. Methods: The in vitro secretion of LH and FSH was measured from 46 cultured NFPAs and compared with pre-operative serum gonadotrophin levels in 38 patients. Peritumorous ‘normal’ pituitary cell cultures from 20 additional pituitary tumour patients were used for comparison with the NFPA group. Results: A median pre-operative LH:FSH ratio of 0.33:1 was found in 38 patients with NFPAs. Preferential secretion of FSH was also documented from media of 46 NFPAs cultured in vitro with a median LH:FSH ratio of 0.32:1. A significant correlation (r = 0.43, P < 0.01) was observed between serum and media levels of FSH but not LH. Peritumorous ‘normal’ pituitary cells released LH and FSH in a reversed ratio (median LH:FSH ratio = 3.6:1, P < 0.01 compared with NFPAs). Conclusions: This study has evaluated pre-operative serum gonadotrophin levels and in vitro release of hormones in cultures of surgically removed tissue from patients with NFPAs. The data suggest preferential secretion of FSH occurs both in vitro and in vivo. By demonstrating that NFPAs cultured in vitro reflect the in vivo situation of preferential secretion of FSH, it may be possible in future to perform functional studies using this system to elucidate the cellular and molecular mechanisms involved in the development of an imbalance in gonadotroph cells preferentially overproducing FSH in NFPAs.

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NPR3 inhibits EMT and Tumour Metastasis of Human Osteosarcoma by Activating AKT/mTOR-mediated Autophagy

10.21203/rs.3.rs-923877/v1 ◽

2021 ◽

Author(s):

Zhihong Yao ◽

Yihao Yang ◽

Jiaxiang Chen ◽

Ting Chen ◽

Lei Han ◽

...

Keyword(s):

Molecular Mechanisms ◽

Therapeutic Potential ◽

Loss Of Function ◽

Over Expression ◽

Functional Studies ◽

Qrt Pcr ◽

Tumour Metastasis ◽

Lower Expression

Abstract BackgroundOsteosarcoma is an aggressive primary malignant cancer of bone mainly occurring in adolescence with a characteristic of high metastasis and relapse rate. In our previous study, we first identified that NPR3 was significantly decreased in OS samples. Here, we purposed to investigate the effect and the possible mechanisms of NPR3 on the progression of human OS. MethodsThe expression of NPR3 in OS patients and cells was detected by qRT-PCR, and IHC analysis. The effect of the expression of NPR3 on tumour metastasis was examined in vitro and in vivo. The molecular mechanisms of the regulation of NPR3 were evaluated in vitro and in vivo. The clinical relevance of 5-year overall survival with the expression of NPR3 was evaluated in 294 patients with OS. ResultsFirstly, we indicated that NPR3 was substantially downregulated expression in OS tissues and cells by qRT-PCR and IHC assay. And the patients with lower expression of NPR3 have a poor prognosis. Functional studies revealed that over-expression of NPR3 inhibited the proliferation and invasion of cells. Meanwhile, over-expression of NPR3 markedly inhibited tumorigenesis and weakened tumour metastasis in vivo. Interestingly, we found that over-expression of NPR3 could induce autophagy, promote apoptosis and inhibit EMT. Additionally, overexpression of NPR3 decreased the phosphorylation levels of AKT and mTOR. Loss-of-function experiments displayed that effects of NPR3 were weakened by treatment with the specific autophagy inhibitor Baf-A1 and CQ. ConclusionsTaken together, these results demonstrated that down-regulation of NPR3 promote lung metastasis of human OS by promoting EMT in part through the AKT/mTOR mediated autophagy, suggesting that NPR3 has therapeutic potential for OS patients with metastasis.

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NLK is required for Ras/ERK/SRF/ELK signaling to tune skeletal muscle development by phosphorylating SRF and antagonizing the SRF/MKL pathway

Cell Death Discovery ◽

10.1038/s41420-021-00774-9 ◽

2022 ◽

Vol 8 (1) ◽

Author(s):

Shang-Ze Li ◽

Ze-Yan Zhang ◽

Jie Chen ◽

Ming-You Dong ◽

Xue-Hua Du ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Muscle Development ◽

Serum Response Factor ◽

Conditional Knockout ◽

Myoblast Differentiation ◽

Knockout Mouse Model ◽

Hypertrophic Growth

AbstractSerum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.

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