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Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF-derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFß and more apoptosis after stimulation with a Fas-activating antibody. Finally, mice with fibroblast-specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Receptor con Dominio Discoidina 2 , Fibrosis Pulmonar Idiopática , Ratones , Animales , Receptor con Dominio Discoidina 2/genética , Receptor con Dominio Discoidina 2/metabolismo , Colágeno Tipo I/metabolismo , Fibroblastos/patología , Colágeno/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Receptores con Dominio Discoidina/metabolismo , Pulmón/patologíaRESUMEN
Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.
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Craneosinostosis , Humanos , Ratones , Animales , Craneosinostosis/genética , Osteogénesis , Linaje de la Célula , Fenotipo , Células MadreRESUMEN
Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.
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Diferenciación Celular , Osteoblastos , Células Madre , Animales , Ratones , Línea Celular , Colágeno/química , Péptidos/química , Péptidos/farmacología , Receptor con Dominio Discoidina 2/química , Integrinas/química , Células Madre/citología , Osteoblastos/citología , Diferenciación Celular/efectos de los fármacos , Fosforilación , Transducción de Señal/efectos de los fármacosRESUMEN
Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in the discoidin domain receptor 2 gene (DDR2), which encodes a non-integrin collagen receptor, are associated with human craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of this gene in craniofacial morphogenesis is not known. As will be shown, Ddr2-deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. These defects were associated with abnormal collagen fibril organization, chondrocyte proliferation and polarization. As established by localization and lineage-tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with GLI1 + cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in GLI +skeletal progenitors and chondrocytes. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor and suggest that DDR2 is necessary for proper collagen organization, chondrocyte proliferation, and orientation.
We each have unique facial features that are key to our identities. These features are inherited, but the mechanisms are poorly understood. People with the genetic disease spondylo-meta-epiphyseal dysplasia, or SMED, have characteristic facial and skull abnormalities including a flattened face and shortened skull. SMED is associated with mutations that inactivate the gene encoding a protein called discoidin domain receptor 2 (DDR2), which is a receptor for collagen. Collagen is the major structural protein in the human body, supporting the structure of cells and tissues. It also controls cell behaviors including growth, migration and differentiation, and it helps form tissues such as cartilage or bone. At least some of the effects of collagen on cells depend on its interaction with DDR2. Since the facial and skull abnormalities in mice with mutations that stop DDR2 from working correctly resemble those of SMED patients, these mice can be used to understand the cellular basis for this disease, as well as the role of DDR2 in the embryonic development of the face and skull. Therefore, Mohamed et al. set out to understand how loss of DDR2 causes the characteristic facial and skull defects associated with SMED. Mohamed et al. used mice that had been genetically modified so that DDR2 could be inactivated in skeletal progenitor cells, cartilage cells and bone cells (osteoblasts). Examining these mice, they found that the shortened skulls and flat face characteristic of mice lacking DDR2 are due to bones at the skull base failing to elongate correctly due to defects in the growth centers that depend on cartilage. Mohamed et al. also discovered that the cells that normally produce DDR2 are the progenitors of cartilage and bone-forming cells, which partly explains why lacking this protein leads to issues in growth of these tissues. In addition to shedding light on the causes of SMED, Mohamed et al.'s results also provide general insights into the mechanisms controlling the formation of facial and skull bones that depend on interactions between cells and collagen. This information may help explain how other abnormalities in the face and skull emerge, and provide a basis for how the shape of the skull has changed during human evolution. In the future, it may be possible to manipulate the activity of DDR2 to correct skull defects.
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Receptor con Dominio Discoidina 2 , Animales , Humanos , Ratones , Cartílago , Condrocitos/fisiología , Colágeno , Receptor con Dominio Discoidina 2/genética , Receptores de ColágenoRESUMEN
The extracellular matrix (ECM) niche plays a critical role in determining cellular behavior during bone development including the differentiation and lineage allocation of skeletal progenitor cells to chondrocytes, osteoblasts, or marrow adipocytes. As the major ECM component in mineralized tissues, collagen has instructive as well as structural roles during bone development and is required for bone cell differentiation. Cells sense their extracellular environment using specific cell surface receptors. For many years, specific ß1 integrins were considered the main collagen receptors in bone, but, more recently, the important role of a second, more primordial collagen receptor family, the discoidin domain receptors, has become apparent. This review will specifically focus on the roles of discoidin domain receptors in mineralized tissue development as well as related functions in abnormal bone formation, regeneration and metabolism.
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Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)-mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.
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Receptor con Dominio Discoidina 2 , Ratones , Animales , Receptor con Dominio Discoidina 2/genética , Proteómica , Diferenciación Celular/genética , Matriz Extracelular/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor kinase that, together with integrins, is required for cells to respond to the extracellular matrix. Ddr2 loss-of-function mutations in humans and mice cause severe defects in skeletal growth and development. However, the cellular functions of Ddr2 in bone are not understood. Expression and lineage analysis showed selective expression of Ddr2 at early stages of bone formation in the resting zone and proliferating chondrocytes and periosteum. Consistent with these findings, Ddr2+ cells could differentiate into hypertrophic chondrocytes, osteoblasts, and osteocytes and showed a high degree of colocalization with the skeletal progenitor marker, Gli1. A conditional deletion approach showed a requirement for Ddr2 in Gli1-positive skeletal progenitors and chondrocytes but not mature osteoblasts. Furthermore, Ddr2 knockout in limb bud chondroprogenitors or purified marrow-derived skeletal progenitors inhibited chondrogenic or osteogenic differentiation, respectively. This work establishes a cell-autonomous function for Ddr2 in skeletal progenitors and cartilage and emphasizes the critical role of this collagen receptor in bone development.
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BACKGROUND: RUNX2, a critical transcription factor in bone development, is also expressed in prostate and breast where it has been linked to cancer progression and cancer stem cells. However, its role in normal prostate biology has not been previously examined. METHODS: Selective growth of murine prostate epithelium under non-adherent conditions was used to enrich for stem cells. Expression of runt domain transcription factors, stem cell and prostate marker messenger RNAs (mRNAs) was determined by quantitative reverse transcription polymerase chain reaction. Effects of Runx2 loss and gain-of-function on prostate epithelial cells were assessed using cells isolated from Runx2loxp/loxp mice transduced with Adeno-Cre or by Adeno-Runx2 transduction of wild type cells. Cellular distribution of RUNX2 and prostate-associated proteins was assessed using immunofluorescence microscopy. In vivo Runx2 knock out was achieved by tamoxifen treatment of Nkx3.1CreERT; Runx2loxp/loxp mice. RESULTS: Prostate epithelium-derived spheroids, which are enriched in stem cells, were shown to contain elevated levels of Runx2 mRNA. Spheroid formation required Runx2 since adenovirus-Cre mediated knockout of Runx2 in prostatic epithelial cells from Runx2loxp/loxp mice severely reduced spheroid formation and stem cell markers while Runx2 overexpression was stimulatory. In vivo, Runx2 was detected during early prostate development (E16.5) and in adult mice where it was present in basal and luminal cells of ventral and anterior lobes. Prostate-selective deletion of Runx2 in tamoxifen-treated Nkx3.1CreERT; Runx2loxp/loxp mice severely inhibited growth and maturation of tubules in the anterior prostate and reduced expression of stem cell markers and prostate-associated genes. CONCLUSION: This study demonstrates an important role for Runx2 in prostate development that may be explained by actions in prostate epithelial stem cells.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Epitelio , Células Madre Neoplásicas/metabolismo , Próstata , Neoplasias de la Próstata , Animales , Antineoplásicos Hormonales/farmacología , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Mutación con Ganancia de Función , Regulación Neoplásica de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Próstata/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero , Tamoxifeno/farmacología , Factores de TranscripciónRESUMEN
Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1+/+ ; Ldlr-/- (single knock-out) and Ddr1-/- ; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.
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Aterosclerosis/enzimología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Angiopatías Diabéticas/enzimología , Receptor con Dominio Discoidina 1/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Calcificación Vascular/enzimología , Transporte Activo de Núcleo Celular , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/patología , Dieta Alta en Grasa , Receptor con Dominio Discoidina 1/deficiencia , Receptor con Dominio Discoidina 1/genética , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Fosforilación , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal , Calcificación Vascular/genética , Calcificación Vascular/patologíaRESUMEN
A common feature of many skeletal diseases is the accumulation of marrow fat. A reciprocal relationship exists between osteogenesis and adipogenesis in bone marrow that is mediated by the relative activity of PPARγ and RUNX2 transcription factors. The ERK/MAPK pathway is an important inducer of MSC differentiation to osteoblasts and an inhibitor of adipogenesis that functions by phosphorylating RUNX2 and PPARγ. To begin to assess the importance of this regulation in vivo, we examined the consequences of blocking one arm of this pathway, PPARγ S112 phosphorylation, by evaluating the bone phenotype of PPARγ S112A mutant mice. This mutation prevents MAPK phosphorylation and inhibition of PPARγ transcriptional activity. Both male and female PPARγ S112A mice had decreased tibial and vertebral BV/TV and decreased trabecular bone relative to wild type littermates. These results were explained by a decrease in bone formation and osteoblast activity in the absence of changes in resorption. In contrast, marrow adipose tissue, adipocyte markers and serum adiponectin were all dramatically increased. Bone marrow stromal cells isolated from PPARγ S112A mice had elevated PPARγ and preferentially differentiated to adipocytes while total and phosphorylated RUNX2 and osteoblastogenesis were inhibited, indicating that the PPARγ S112A mutation affects bone in a cell autonomous manner. Changes in osteoblast/adipocyte lineage allocation in MSC cultures were also seen where CFU-OBs were reduced with a parallel increase in CFU-AD. This study emphasizes the importance of PPARγ phosphorylation in controlling bone mass and marrow adiposity and demonstrates how a regulatory mutation in PPARγ previously associated with peripheral fat metabolism can have broader effects on bone homeostasis that may in turn affect whole body energy metabolism.
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Adipogénesis/fisiología , Médula Ósea/fisiología , Osteogénesis/fisiología , PPAR gamma/metabolismo , Animales , Diferenciación Celular/fisiología , Metabolismo Energético/fisiología , Femenino , Homeostasis/fisiología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , FosforilaciónRESUMEN
PURPOSE OF THE REVIEW: This review will provide a timely assessment of MAP kinase actions in bone development and homeostasis with particular emphasis on transcriptional control of the osteoblast lineage. RECENT FINDINGS: ERK and p38 MAP kinases function as transducers of signals initiated by the extracellular matrix, mechanical loading, TGF-ß, BMPs and FGF2. MAPK signals may also affect and/or interact with other important pathways such as WNT and HIPPO. ERK and p38 MAP kinase pathways phosphorylate specific osteogenic transcription factors including RUNX2, Osterix, ATF4 and DLX5. For RUNX2, phosphorylation at specific serine residues initiates epigenetic changes in chromatin necessary for decondensation and increased transcription. MAPK also suppresses marrow adipogenesis by phosphorylating and inhibiting PPARγ, which may explain the well-known relationship between reduced skeletal loading and marrow fat accumulation. SUMMARY: MAPKs transduce signals from the extracellular environment to the nucleus allowing bone cells to respond to changes in hormonal/growth factor signaling and mechanical loading thereby optimizing bone structure to meet physiological and mechanical needs of the body.
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Increased collagen deposition by breast cancer (BC)-associated mesenchymal stem/multipotent stromal cells (MSC) promotes metastasis, but the mechanisms are unknown. Here, we report that the collagen receptor discoidin domain receptor 2 (DDR2) is essential for stromal-BC communication. In human BC metastasis, DDR2 is concordantly upregulated in metastatic cancer and multipotent mesenchymal stromal cells. In MSCs isolated from human BC metastasis, DDR2 maintains a fibroblastic phenotype with collagen deposition and induces pathological activation of DDR2 signaling in BC cells. Loss of DDR2 in MSCs impairs their ability to promote DDR2 phosphorylation in BC cells, as well as BC cell alignment, migration, and metastasis. Female ddr2-deficient mice homozygous for the slie mutation show inefficient spontaneous BC metastasis. These results point to a role for mesenchymal stem cell DDR2 in metastasis and suggest a therapeutic approach for metastatic BC.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/fisiología , Receptor con Dominio Discoidina 2/metabolismo , Células Madre Mesenquimatosas/metabolismo , Metástasis de la Neoplasia/patología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Colágeno/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Fosforilación/fisiología , Receptores de Colágeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase-dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2-binding regions of Bglap2 and Ibsp. Growth of MC3T3-E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin-bound P-ERK, P-RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di-methylation, another gene activation-associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono-, di-, and tri-methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation-dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2-deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non-phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin-associated P-ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P-RUNX2-dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427-2435, 2017. © 2016 Wiley Periodicals, Inc.
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Diferenciación Celular , Ensamble y Desensamble de Cromatina , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Epigénesis Genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/enzimología , Osteogénesis , Células 3T3 , Acetilación , Animales , Sitios de Unión , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/genética , Metilación de ADN , Regulación de la Expresión Génica , Histonas/metabolismo , Sialoproteína de Unión a Integrina/genética , Sialoproteína de Unión a Integrina/metabolismo , Metilación , Ratones , Mutación , Osteogénesis/genética , Fosforilación , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Peroxisome proliferator-activated receptor γ (PPARγ) and runt-related transcription factor 2 (RUNX2) are key regulators of mesenchymal stem cell (MSC) differentiation toward adipocytes and osteoblasts, respectively. Post-translational modifications of these factors determine their activities. Dephosphorylation of PPARγ at Ser-112 is required for its adipocytic activity, whereas phosphorylation of RUNX2 at serine 319 (Ser-319) promotes its osteoblastic activity. Here we show that protein phosphatase 5 (PP5) reciprocally regulates each receptor by targeting each serine. Mice deficient in PP5 phosphatase have increased osteoblast numbers and high bone formation, which results in high bone mass in the appendicular and axial skeleton. This is associated with a substantial decrease in lipid-containing marrow adipocytes. Indeed, in the absence of PP5 the MSC lineage allocation is skewed toward osteoblasts and away from lipid accumulating adipocytes, although an increase in beige adipocyte gene expression is observed. In the presence of rosiglitazone, PP5 translocates to the nucleus, binds to PPARγ and RUNX2, and dephosphorylates both factors, resulting in activation of PPARγ adipocytic and suppression of RUNX2 osteoblastic activities. Moreover, shRNA knockdown of PP5 results in cells refractory to rosiglitazone treatment. Lastly, mice deficient in PP5 are resistant to the negative effects of rosiglitazone on bone, which in wild type animals causes a 50% decrease in trabecular bone mass. In conclusion, PP5 is a unique phosphatase reciprocally regulating PPARγ and RUNX2 activities in marrow MSC.
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Peso Corporal/efectos de los fármacos , Huesos/metabolismo , Núcleo Celular/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Glicoproteínas/metabolismo , PPAR gamma/metabolismo , Tiazolidinedionas/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Animales , Peso Corporal/genética , Núcleo Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Glicoproteínas/genética , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , PPAR gamma/genética , RosiglitazonaRESUMEN
Cell-extracellular matrix (ECM) interactions play major roles in controlling progenitor cell fate and differentiation. The receptor tyrosine kinase, discoidin domain receptor 2 (DDR2), is an important mediator of interactions between cells and fibrillar collagens. DDR2 signals through both ERK1/2 and p38 MAP kinase, which stimulate osteoblast differentiation and bone formation. Here we show that DDR2 is critical for skeletal development and differentiation of marrow progenitor cells to osteoblasts while suppressing marrow adipogenesis. Smallie mice (Ddr2slie/slie ), which contain a nonfunctional Ddr2 allele, have multiple skeletal defects. A progressive decrease in tibial trabecular bone volume/total volume (BV/TV) was observed when wild-type (WT), Ddr2wt/slie , and Ddr2slie/slie mice were compared. These changes were associated with reduced trabecular number (Tb.N) and trabecular thickness (Tb.Th) and increased trabecular spacing (Tb.Sp) in both males and females, but reduced cortical thickness only in Ddr2slie/slie females. Bone changes were attributed to decreased bone formation rather than increased osteoclast activity. Significantly, marrow fat and adipocyte-specific mRNA expression were significantly elevated in Ddr2slie/slie animals. Additional skeletal defects include widened calvarial sutures and reduced vertebral trabecular bone. To examine the role of DDR2 signaling in cell differentiation, bone marrow stromal cells (BMSCs) were grown under osteogenic and adipogenic conditions. Ddr2slie/slie cells exhibited defective osteoblast differentiation and accelerated adipogenesis. Changes in differentiation were related to activity of runt-related transcription factor 2 (RUNX2) and PPARγ, transcription factors that are both controlled by MAPK-dependent phosphorylation. Specifically, the defective osteoblast differentiation in calvarial cells from Ddr2slie/slie mice was associated with reduced ERK/MAP kinase and RUNX2-S319 phosphorylation and could be rescued with a constitutively active phosphomimetic RUNX2 mutant. Also, DDR2 was shown to increase RUNX2-S319 phosphorylation and transcriptional activity while also increasing PPARγ-S112 phosphorylation, but reducing its activity. DDR2 is, therefore, important for maintenance of osteoblast activity and suppression of marrow adipogenesis in vivo and these actions are related to changes in MAPK-dependent RUNX2 and PPARγ phosphorylation. © 2016 American Society for Bone and Mineral Research.
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Adipogénesis , Médula Ósea/metabolismo , Receptor con Dominio Discoidina 2/metabolismo , Osteogénesis , Adiposidad , Animales , Células COS , Calcificación Fisiológica , Hueso Esponjoso/patología , Chlorocebus aethiops , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , PPAR gamma/metabolismo , Fosforilación , Cráneo/patologíaRESUMEN
In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ-dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant-negative suppression of activity had the opposite effect. In rescue experiments using Runx2(-/-) mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation-deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation-resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg(-/-) MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages.