Impaired autophagy triggered by HDAC9 in mesenchymal stem cells accelerates bone mass loss

Abstract Background: Bone mass loss in aging is linked with imbalanced lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs). Recent studies have proved that histone deacetylases (HDACs) are regarded as key regulators of bone remodeling. However, HDACs involve in regulating BMMSCs bio-behaviors remain elusive. Here, we investigated ability of HDAC9 on modulation of autophagy and its significance in lineage differentiation of BMMSCs.Methods: The effects of HDAC9 on lineage differentiation of BMMSCs and autophagic signaling were assessed by various biochemical (Western blot and ChIP assay), morphological (TEM and confocal microscopy) and microCT assays.Results ：16-month mice manifested obvious bone mass loss and marrow fat increase, accompanied with decreased osteogenic differentiation and increased adipogenic differentiation of BMMSCs. Further, the expression of HDAC9 elevated in bone and BMMSCs. Importantly, HDAC9 inhibitors recovered the lineage differentiation abnormality of 16-month BMMSCs and reduced p53 expression. Mechanistically, we revealed that HDAC9 regulated the autophagy of BMMSCs by controlling H3K9 acetylation in the promoters of the autophagic genes, ATG7 , BECN1 , and LC3a/b, which subsequently affected their lineage differentiation. Finally, HDAC9 inhibition improved endogenous BMMSCs properties and promoted the bone mass recovery of 16-month mice.Conclusions: Our data demonstrate that HDAC9 is a key regulator in variety of bone mass by regulating autophagic activity in BMMSCs and thus a potential target of age-related bone loss treatment.

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Impaired autophagy triggered by HDAC9 in mesenchymal stem cells accelerates bone mass loss

10.21203/rs.3.rs-20744/v1 ◽

2020 ◽

Author(s):

Liqiang Zhang ◽

Meng Qi ◽

Ji Chen ◽

Jiangdong Zhao ◽

Liya Li ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Mass ◽

Mass Loss ◽

Adipogenic Differentiation ◽

Marrow Fat ◽

P53 Expression ◽

Bone Mass Loss ◽

Lineage Differentiation ◽

Age Related

Abstract Background: Bone mass loss in aging is linked with imbalanced lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs). Recent studies have proved that histone deacetylases (HDACs) are regarded as key regulators of bone remodeling. However, HDACs involve in regulating BMMSCs bio-behaviors remain elusive. Here, we investigated ability of HDAC9 on modulation of autophagy and its significance in lineage differentiation of BMMSCs. Methods: The effects of HDAC9 on lineage differentiation of BMMSCs and autophagic signalling were assessed by various biochemical (Western blot and ChIP assay), morphological (TEM and confocal microscopy) and microCT assays. Results：16-month mice manifested obvious bone mass loss and marrow fat increase, accompanied with the decreased osteogenic differentiation and increased adipogenic differentiation of BMMSCs. Further, the expression of HDAC9 elevated in bone and BMMSCs. Importantly, HDAC9 inhibitors recovered the lineage differentiation of 16-month BMMSCs and reduced p53 expression. Mechanistically, we revealed that HDAC9 regulated the autophagy of BMMSCs by controlling H3K9 acetylation in the promoters of the autophagic genes, ATG7 , BECN1 , and LC3a/b, which subsequently affected their lineage differentiation. Finally, HDAC9 inhibition improved endogenous BMMSCs properties and promoted the bone mass recovery of 16-month mice. Conclusions: Our data demonstrate that HDAC9 is a key regulator in variety of bone mass by regulating autophagic activity in BMMSCs and is thus, a potential target of age-related bone loss treatment.

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Impaired autophagy triggered by HDAC9 in mesenchymal stem cells accelerates bone mass loss

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01785-6 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Liqiang Zhang ◽

Meng Qi ◽

Ji Chen ◽

Jiangdong Zhao ◽

Liya Li ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Mass ◽

Mass Loss ◽

Bone Mass Loss

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The Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in Human Suprapatellar Fat Pad Derived Mesenchymal Stem Cells

Molecules ◽

10.3390/molecules26071831 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1831

Author(s):

Thitianan Kulsirirat ◽

Sittisak Honsawek ◽

Mariko Takeda-Morishita ◽

Nuttanan Sinchaipanid ◽

Wanvisa Udomsinprasert ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Fat Pad ◽

Alizarin Red ◽

Lineage Differentiation ◽

Expression Of Genes ◽

Dose Dependent ◽

Oil Red O Staining

Andrographolide is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculata, and widely used for its potential medical properties. However, there are no reports on the effects of andrographolide on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of andrographolide on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. The results revealed that andrographolide had no cytotoxic effects when the concentration was less than 12.5 µM. Interestingly, andrographolide had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, andrographolide can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, andrographolide suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. These findings confirm that andrographolide can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.

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Bromodomain Protein BRD4 Accelerates Glucocorticoid Dysregulation of Bone Mass and Marrow Adiposis by Modulating H3K9 and Foxp1

Cells ◽

10.3390/cells9061500 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1500

Author(s):

Feng-Sheng Wang ◽

Yu-Shan Chen ◽

Jih-Yang Ko ◽

Chung-Wen Kuo ◽

Huei-Jing Ke ◽

...

Keyword(s):

Bone Mass ◽

Mass Loss ◽

Osteogenic Differentiation ◽

Progenitor Cells ◽

Mesenchymal Progenitor Cells ◽

Marrow Fat ◽

Bone Mass Loss ◽

Brd4 Inhibition ◽

The Cost

Glucocorticoid provokes bone mass loss and fatty marrow, accelerating osteoporosis development. Bromodomain protein BRD4, an acetyl–histone-binding chromatin reader, regulates stem cell and tissue homeostasis. We uncovered that glucocorticoid inhibited acetyl Lys-9 at the histone 3 (H3K9ac)-binding Runx2 promoter and decreased osteogenic differentiation, whereas bromodomain protein 4 (BRD4) and adipocyte formation were upregulated in bone-marrow mesenchymal progenitor cells. BRD4 knockdown improved H3K9ac occupation at the Runx2 promoter and osteogenesis, but attenuated glucocorticoid-mediated adipocyte formation together with the unaffected H3K9ac-binding PPARγ2 promoter. BRD4 regulated epigenome related to fatty acid metabolism and the forkhead box P1 (Foxp1) pathway, which occupied the PPARγ2 promoter to modulate glucocorticoid-induced adipocytic activity. In vivo, BRD4 inhibitor JQ-1 treatment mitigated methylprednisolone-induced suppression of bone mass, trabecular microstructure, mineral acquisition, and osteogenic differentiation. Foxp1 signaling, marrow fat, and adipocyte formation in glucocorticoid-treated skeleton were reversed upon JQ-1 treatment. Taken together, glucocorticoid-induced H3K9 hypoacetylation augmented BRD4 action to Foxp1, which steered mesenchymal progenitor cells toward adipocytes at the cost of osteogenic differentiation in osteoporotic skeletons. BRD4 inhibition slowed bone mass loss and marrow adiposity. Collective investigations convey a new epigenetic insight into acetyl histone reader BRD4 control of osteogenesis and adipogenesis in skeleton, and highlight the remedial effects of the BRD4 inhibitor on glucocorticoid-induced osteoporosis.

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Mechanisms Underlying the Osteo- and Adipo-Differentiation of Human Mesenchymal Stem Cells

The Scientific World JOURNAL ◽

10.1100/2012/793823 ◽

2012 ◽

Vol 2012 ◽

pp. 1-14 ◽

Cited By ~ 58

Author(s):

Yu Zhang ◽

Dilaware Khan ◽

Julia Delling ◽

Edda Tobiasch

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Human Mscs ◽

Industrialized Countries ◽

Tight Control ◽

Age Related ◽

Donor Cells ◽

Immunosuppressive Microenvironment

Human mesenchymal stem cells (hMSCs) are considered a promising cell source for regenerative medicine, because they have the potential to differentiate into a variety of lineages among which the mesoderm-derived lineages such adipo- or osteogenesis are investigated best. Human MSCs can be harvested in reasonable to large amounts from several parts of the patient’s body and due to this possible autologous origin, allorecognition can be avoided. In addition, even in allogenic origin-derived donor cells, hMSCs generate a local immunosuppressive microenvironment, causing only a weak immune reaction. There is an increasing need for bone replacement in patients from all ages, due to a variety of reasons such as a new recreational behavior in young adults or age-related diseases. Adipogenic differentiation is another interesting lineage, because fat tissue is considered to be a major factor triggering atherosclerosis that ultimately leads to cardiovascular diseases, the main cause of death in industrialized countries. However, understanding the differentiation process in detail is obligatory to achieve a tight control of the process for future clinical applications to avoid undesired side effects. In this review, the current findings for adipo- and osteo-differentiation are summarized together with a brief statement on first clinical trials.

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The Influence of Aging on the Regenerative Potential of Human Adipose Derived Mesenchymal Stem Cells

Stem Cells International ◽

10.1155/2016/2152435 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 99

Author(s):

Monika Marędziak ◽

Krzysztof Marycz ◽

Krzysztof A. Tomaszewski ◽

Katarzyna Kornicka ◽

Brandon Michael Henry

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Population Doubling ◽

Osteogenic Potential ◽

Population Doubling Time ◽

Joint Diseases ◽

Differentiation Potential ◽

Age Related

Tissue regeneration using human adipose derived mesenchymal stem cells (hASCs) has significant potential as a novel treatment for many degenerative bone and joint diseases. Previous studies have established that age negatively affects the proliferation status and the osteogenic and chondrogenic differentiation potential of mesenchymal stem cells. The aim of this study was to assess the age-related maintenance of physiological function and differentiation potential of hASCs in vitro. hASCs were isolated from patients of four different age groups: (1) >20 years (n=7), (2) >50 years (n=7), (3) >60 years (n=7), and (4) >70 years (n=7). The hASCs were characterized according to the number of fibroblasts colony forming unit (CFU-F), proliferation rate, population doubling time (PDT), and quantified parameters of adipogenic, chondrogenic, and osteogenic differentiation. Compared to younger cells, aged hASCs had decreased proliferation rates, decreased chondrogenic and osteogenic potential, and increased senescent features. A shift in favor of adipogenic differentiation with increased age was also observed. As many bone and joint diseases increase in prevalence with age, it is important to consider the negative influence of age on hASCs viability, proliferation status, and multilineage differentiation potential when considering the potential therapeutic applications of hASCs.

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Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

10.1101/2021.11.29.470107 ◽

2021 ◽

Author(s):

Kannan Govindaraj ◽

Sakshi Khurana ◽

Marcel Karperien ◽

Janine Nicole Post

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Transcription Factor ◽

Single Cell ◽

Adipogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Cell Types ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Transcriptional Dynamics

The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about the signaling interplay during differentiation remains one of the main reasons preventing successful application of hMSCs in regenerative medicine. We previously showed that Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) can be used to study SOX9 dynamics at the single cell level. We showed that changes in SOX9 dynamics are linked to its transcriptional activity. Here, we investigated SOX9 dynamics during differentiation of hMSCs into the chondrogenic, osteogenic and adipogenic lineages. We show that there are clusters of cells in hMSCs with distinct SOX9 dynamics, indicating that there are a number of subpopulations present in the heterogeneous hMSCs. SOX9 dynamics data at the single cell resolution revealed novel insights about its activity in these subpopulations (cell types). In addition, the response of SOX9 to differentiation stimuli varied in these subpopulations. Moreover, we identified donor specific differences in the number of cells per cluster in undifferentiated hMSCs, and this correlated to their differentiation potential.

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Phactr1 Negatively Regulates Bone Mass By Inhibiting Osteogenesis And Promoting Adipogenesis of BMSCs Via RhoA/ROCK2

10.21203/rs.3.rs-820284/v1 ◽

2021 ◽

Author(s):

Wei Lin ◽

Zhipeng Chen ◽

Xiaoyi Mo ◽

Shengli Zhao ◽

Zhenxing Wen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Bone Mass ◽

Osteogenic Differentiation ◽

Adipogenic Differentiation ◽

Regulatory Factor

Abstract Background: The imbalance between osteogenic and adipogenic differentiation of Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) is involved in the occurrence and development of Osteoporosis (OP). Previous studies have indicated the potential of phosphatase and actin regulatory factor 1 (Phactr1) in regulating osteogenic and adipogenic differentiation of BMSCs.The present study aims to investigate The Function and Mechanism of Phactr1 in regulating osteogenic and adipogenic Differentiation of BMSCs.Results: Phactr1 increased in both bone and adipose tissue of OP rats. During osteogenic differentiation , Phactr1 decreased and active RhoA, ROCK2 increased, while overexpression Phactr1 inhibits the increase of Runx2. Phactr1 increased and active RhoA decreased, ROCK2 did not changed during adipogenic differentiation, knockdown Phactr1 inhibits the increase of C/EBPα. Phactr1 and ROCK2 were combined in osteogenic differentiation, but not in adipogenic differentiation. By using KD025, the decrease of Phactr1 and the increase of Runx2 were inhibited respectively in osteogenic differentiation. Meanwhile, when ROCK2 was inhibited, Phactr1,C/EBPα were significantly increased in adipogenic differentiation.Conclusions: These findings indicated that Phactr1 negatively regulates bone mass by inhibiting osteogenesis and promoting adipogenesis of BMSCs by activating RhoA/ROCK2.

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Ionomycin Ameliorates Hypophosphatasia via Rescuing Alkaline Phosphatase Deficiency-mediated L-type Ca2+ Channel Internalization in Mesenchymal Stem Cells

10.1101/545418 ◽

2019 ◽

Author(s):

Bei Li ◽

Xiaoning He ◽

Zhiwei Dong ◽

Kun Xuan ◽

Wei Sun ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Cell Lineage ◽

Bone Homeostasis ◽

Loss Of Function ◽

Channel Trafficking ◽

Lineage Differentiation ◽

Promising Therapeutic Approach ◽

Intracellular Ca

AbstractLoss-of-function mutations in ALPL result in hypophosphatasia (HPP), an inborn error of metabolism that causes skeletal mineralization defect. In adults, main clinical involvement includes early loss of primary or secondary teeth, osteoporosis, bone pain, chondrocalcinosis, and fractures. However, guidelines for the treatment of adults with HPP are not available. Here, we show that ALPL deficiency caused reduction of intracellular Ca2+ influx resulting in osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse BMSCs. To elevate intracellular level of calcium in bone marrow mesenchymal stem cells (BMSCs) by ionomycin treatment rescues the osteoporotic phenotype in alpl+/- mice and BMSC-specific (Prrx1-alpl-/-) conditional alpl knockout mice. Mechanistically, ALPL is required to maintain intracellular Ca2+ influx by regulating L-type Ca2+ channel trafficking via binding to the α2δ subunits, which regulates the internalization of L-type Ca2+ channel. Decreased Ca2+ flux inactivates Akt/GSK3β/β-catenin signaling pathway that regulates lineage differentiation of BMSCs. This study identifies a previous unknown role of ectoenzyme ALPL in maintenance of calcium channel trafficking to keep stem cell lineage differentiation and bone homeostasis. Accelerating Ca2+ flux through L-type Ca2+ channel by ionomycin treatment may be a promising therapeutic approach for adult HPP patients.One Sentence SummaryALP regulates internalization of L-Type Ca2+ Channel of BMSCs in Hypophosphatasia.

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Increased BMPR1A Expression Enhances the Adipogenic Differentiation of Mesenchymal Stem Cells in Patients with Ankylosing Spondylitis

Stem Cells International ◽

10.1155/2019/4143167 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Zhenhua Liu ◽

Peng Wang ◽

Shuizhong Cen ◽

Liangbin Gao ◽

Zhongyu Xie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Ankylosing Spondylitis ◽

Signaling Pathway ◽

Adipogenic Differentiation ◽

Marrow Fat ◽

Differentiation Capacity ◽

Bone Marrow Fat ◽

Healthy Donors

Objective. To investigate the adipogenic differentiation capacity of mesenchymal stem cells (MSCs) from ankylosing spondylitis (AS) patients and explore the mechanism of abnormal MSC adipogenesis in AS. Methods. MSCs from patients with AS (ASMSCs) and healthy donors (HDMSCs) were cultured in adipogenic differentiation medium for up to 21 days. Adipogenic differentiation was determined using oil red O (ORO) staining and quantification and was confirmed by assessing adipogenic marker expression (PPAR-γ, FABP4, and adiponectin). Gene expression of adipogenic markers was detected using qRT-PCR. Protein levels of adipogenic markers and signaling pathway-related molecules were assessed via Western blotting. Levels of bone morphogenetic proteins 4, 6, 7, and 9 were determined using enzyme-linked immunosorbent assays. Lentiviruses encoding short hairpin RNAs (shRNAs) were constructed to reverse abnormal bone morphogenetic protein receptor 1A (BMPR1A) expression and evaluate its role in abnormal ASMSC adipogenic differentiation. Bone marrow fat content was assessed using hematoxylin and eosin (HE) staining. BMPR1A expression in bone marrow MSCs was measured using immunofluorescence staining. Results. ASMSCs exhibited a greater adipogenic differentiation capacity than HDMSCs. During adipogenesis, ASMSCs expressed BMPR1A at higher levels, which activated the BMP-pSmad1/5/8 signaling pathway and increased adipogenesis. BMPR1A silencing using an shRNA eliminated the difference in adipogenic differentiation between HDMSCs and ASMSCs. Moreover, HE and immunofluorescence staining showed higher bone marrow fat content and BMPR1A expression in patients with AS than in healthy donors. Conclusion. Increased BMPR1A expression induces abnormal ASMSC adipogenic differentiation, potentially contributing to fat metaplasia and thus new bone formation in patients with AS.

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