The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stromal /stem cells through interacting with the Notch1 protein

Abstract Background Immunomodulatory activities of human mesenchymal stromal /stem cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before. Methods The transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4+-Th1 lymphocytes, and the release of immunomodulatory molecule IDO1. Statistical analysis One-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments. Results The present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs. Conclusions The present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.

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The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stromal /stem cells through interacting with the Notch1 protein

10.21203/rs.2.19213/v4 ◽

2020 ◽

Author(s):

Tao Na ◽

Kehua Zhang ◽

Bao-Zhu Yuan

Keyword(s):

Stem Cells ◽

Tumor Suppressor ◽

Protein Degradation ◽

Signaling Pathway ◽

Mutual Exclusion ◽

Fine Tuning ◽

Therapeutic Effects ◽

Notch1 Signaling ◽

Stromal Stem Cells ◽

Immunomodulatory Function

Abstract BackgroundImmunomodulatory activities of human mesenchymal stromal /stem cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before.MethodsThe transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4+-Th1 lymphocytes, and the release of immunomodulatory molecule IDO1.Statistical analysisOne-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments.ResultsThe present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs.ConclusionsThe present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.

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The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stem cells through interacting with the Notch1 protein

10.21203/rs.2.19213/v2 ◽

2020 ◽

Author(s):

Tao Na ◽

Kehua Zhang ◽

Bao-Zhu Yuan

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Tumor Suppressor ◽

Protein Degradation ◽

Signaling Pathway ◽

Mutual Exclusion ◽

Human Mesenchymal Stem Cells ◽

Fine Tuning ◽

Notch1 Signaling ◽

Immunomodulatory Function

Abstract Background Immunomodulatory activities of human mesenchymal stem cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before. Methods The transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4+-Th1 lymphocytes, and the release of immunomodulatory molecule IDO1. Statistical analysis One-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments. Results The present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs. Conclusions The present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.

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The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stem/stromal cells through interacting with the Notch1 protein

10.21203/rs.2.19213/v3 ◽

2020 ◽

Author(s):

Tao Na ◽

Kehua Zhang ◽

Bao-Zhu Yuan

Keyword(s):

Tumor Suppressor ◽

Protein Degradation ◽

Signaling Pathway ◽

Stromal Cells ◽

Mutual Exclusion ◽

Fine Tuning ◽

Therapeutic Effects ◽

E3 Ligases ◽

Notch1 Signaling ◽

Immunomodulatory Function

Abstract Background Immunomodulatory activities of human mesenchymal stem/stromal cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before. Methods The transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4 + -Th1 lymphocytes, and the release of immunomodulatory molecule IDO1. Statistical analysis One-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments. Results The present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs. Conclusions The present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.

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The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stem cells through interacting with the Notch1 protein

10.21203/rs.2.19213/v1 ◽

2019 ◽

Author(s):

Tao Na ◽

Kehua Zhang ◽

Bao-Zhu Yuan

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Tumor Suppressor ◽

Mutual Exclusion ◽

Human Mesenchymal Stem Cells ◽

Fine Tuning ◽

Therapeutic Effects ◽

Critical Function ◽

E3 Ligases ◽

Notch1 Signaling

Abstract Immunomodulatory activities of human mesenchymal stem cells (hMSCs) have been widely accepted as the most critical function of the cells for exerting its therapeutic effects. The activities include the inhibition by hMSCs on pro-inflammatory CD4 + -T lymphocytes, and the release of immunomodulatory molecules, like IDO1. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previously, the Notch1 protein has been demonstrated to be able to promote the immunomodulation of hMSCs through inhibiting CD4 + -Th1 lymphocyte proliferation and enhancing IDO1 expression. The present study further revealed that it was the Notch1-Hey1 axis, rather than the Notch1-Hes1 axis, that was likely responsible for mediating the immunomodulation of the Notch1 signaling. Meanwhile, following a previously proposed hypothesis to identify proteasome-regulated protein(s) for limiting the activity of the Notch1 signaling in hMSCS, the DLC-1 tumor suppressor was identified to be such a candidate protein, which was subjected to protein degradation mediated by the DDB1 and FBXW5 E3 ligases . It was further shown that the DLC-1 signaling composing of DLC-1, Rock1 and FBXW5 proteins was involved in inhibiting the immunomodulation of hMSCs. More importantly, the immunomodulation was achieved through an interaction between the DLC-1-FBXW5-Rock1 signaling and the Notch1-Hey1 signaling . In fact, the present study a novel function of DLC-1 tumor suppressor as well as proposed a new mutual exclusion mechanism likely responsible for fine-tuning the immunomodulation of hMSCs.

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Neonatal Anesthesia by Ketamine in Neonatal Rats Inhibits the Proliferation and Differentiation of Hippocampal Neural Stem Cells and Decreases Neurocognitive Function in Adulthood via Inhibition of the Notch1 Signaling Pathway

Molecular Neurobiology ◽

10.1007/s12035-021-02550-3 ◽

2021 ◽

Author(s):

He Huang ◽

Chao Zhao ◽

Qian Hu ◽

Qiang Liu ◽

Yi-Man Sun ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Signaling Pathway ◽

Neurocognitive Function ◽

Neonatal Rats ◽

Notch1 Signaling ◽

Proliferation And Differentiation ◽

Notch1 Signaling Pathway ◽

Hippocampal Neural Stem Cells

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Cancer Stem Cells, Quo Vadis? The Notch Signaling Pathway in Tumor Initiation and Progression

Cells ◽

10.3390/cells9081879 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1879 ◽

Cited By ~ 2

Author(s):

Christian T. Meisel ◽

Cristina Porcheri ◽

Thimios A. Mitsiadis

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Notch Signaling ◽

Signaling Pathway ◽

Cell Fate ◽

Adult Life ◽

Notch Pathway ◽

Notch Signaling Pathway ◽

Fine Tuning ◽

Cell Fate Decisions

The Notch signaling pathway regulates cell proliferation, cytodifferentiation and cell fate decisions in both embryonic and adult life. Several aspects of stem cell maintenance are dependent from the functionality and fine tuning of the Notch pathway. In cancer, Notch is specifically involved in preserving self-renewal and amplification of cancer stem cells, supporting the formation, spread and recurrence of the tumor. As the function of Notch signaling is context dependent, we here provide an overview of its activity in a variety of tumors, focusing mostly on its role in the maintenance of the undifferentiated subset of cancer cells. Finally, we analyze the potential of molecules of the Notch pathway as diagnostic and therapeutic tools against the various cancers.

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Mesenchymal Stromal/Stem Cells in Regenerative Medicine and Tissue Engineering

Stem Cells International ◽

10.1155/2018/8031718 ◽

2018 ◽

Vol 2018 ◽

pp. 1-16 ◽

Cited By ~ 96

Author(s):

Ross E. B. Fitzsimmons ◽

Matthew S. Mazurek ◽

Agnes Soos ◽

Craig A. Simmons

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Regenerative Medicine ◽

Ex Vivo ◽

Therapeutic Effects ◽

Wide Range ◽

History Of ◽

Initial Discovery ◽

Stromal Stem Cells

As a result of over five decades of investigation, mesenchymal stromal/stem cells (MSCs) have emerged as a versatile and frequently utilized cell source in the fields of regenerative medicine and tissue engineering. In this review, we summarize the history of MSC research from the initial discovery of their multipotency to the more recent recognition of their perivascular identity in vivo and their extraordinary capacity for immunomodulation and angiogenic signaling. As well, we discuss long-standing questions regarding their developmental origins and their capacity for differentiation toward a range of cell lineages. We also highlight important considerations and potential risks involved with their isolation, ex vivo expansion, and clinical use. Overall, this review aims to serve as an overview of the breadth of research that has demonstrated the utility of MSCs in a wide range of clinical contexts and continues to unravel the mechanisms by which these cells exert their therapeutic effects.

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UBA2 Activates Wnt/ β-catenin Signaling Pathway during Protection of R28 Retinal Precursor Cells from Hypoxia by Extracellular Vesicles derived from Placental Mesenchymal Stem Cells

10.21203/rs.3.rs-33694/v3 ◽

2020 ◽

Author(s):

Kyungmin Koh ◽

Mira Park ◽

Eun Soo Bae ◽

Van-An Duong ◽

Jong-Moon Park ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Treatment Group ◽

Signaling Pathway ◽

Precursor Cells ◽

Therapeutic Effects ◽

Label Free ◽

Related Factors ◽

Cell Therapies ◽

Placental Mesenchymal Stem Cells

Abstract Background: Stem cell transplantation has been proposed as an alternative treatment for intractable optic nerve disorders characterized by irrecoverable loss of cells. Mesenchymal stem cells, with varying tissue regeneration and recovery capabilities, are being considered for potential cell therapies. To overcome the limitations of cell therapy, we isolated exosomes from human placenta–derived mesenchymal stem cells (hPMSCs), and investigated their therapeutic effects in R28 cells (retinal precursor cells) exposed to CoCl2. Method: After nine hours of exposure to CoCl2, the hypoxic damaged R28 cells were divided into non treatment group (CoCl2+R28 cells) and treatment group (CoCl2+R28 cells treated with exosome). Immunoblot analysis was performed for Pcna, Hif-1α, Vegf, Vimentin, Thy-1, Gap43, Ermn, Neuroflament, Wnt3a, β-catenin, phospo-GSK3β, Lef-1, UBA2, Skp1, βTrcp, and ubiquitin. The proteomes of each group were analyzed by liquid chromatography/tandem mass (LC-MS/MS) spectrometry. Differentially expressed proteins (DEPs) were detected by label-free quantification and the interactions of the proteins were examined through signal transduction pathway and gene ontology analysis. Result: We observed that Exosome could significantly recover proliferation damaged by CoCl2 treatment. In addition, treatment group presented the decreased expression of Hif-1α protein (P < 0.05) and increased expression of proliferation marker, Pcna, and nerve regeneration–related factors such as Vimentin, Thy-1 and Neuroflament (P < 0.05) compared with non-treatment group. In total, 200 DEPs were identified in non-treatment group and treatment group (fold change ≥ 2, p < 0.05). Catenin and ubiquitin systems (UBA2, UBE2E3, UBE2I) were found in both the DEP lists of downregulated proteins from non-treatment group and upregulated proteins from treatment group. The mRNA expressions of ubiquitin systems were significantly decreased under hypoxic condition. Moreover, UBA2 and Wnt/β-catenin protein were associated with rescue of the hypoxic damaged R28 cells. Using a siRNA system, we could find it out that hPMSC exosoms could not repair altered expressions of target proteins by CoCl2 in lacking UBA2 R28 cells. Conclusion: This study reported that hypoxic damaged expression of regeneration markers in R28 cells were significantly recovered by hPMSC exosomes. We could also demonstrate that UBA2 played a key role in activating the Wnt/β-catenin signaling pathway during protection of hypoxic damaged R28 cells, induced by hPMSC exosomes.

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Changes in the Transcriptome Profiles of Human Amnion-Derived Mesenchymal Stromal/Stem Cells Induced by Three-Dimensional Culture: A Potential Priming Strategy to Improve Their Properties

International Journal of Molecular Sciences ◽

10.3390/ijms23020863 ◽

2022 ◽

Vol 23 (2) ◽

pp. 863

Author(s):

Alessia Gallo ◽

Nicola Cuscino ◽

Flavia Contino ◽

Matteo Bulati ◽

Mariangela Pampalone ◽

...

Keyword(s):

Stem Cells ◽

Three Dimensional ◽

3D Culture ◽

Therapeutic Effects ◽

Rna Seq ◽

Human Amnion ◽

Cell Based Therapy ◽

Therapeutic Properties ◽

Stromal Stem Cells

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.

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