RESUMEN

Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan (GAG) rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address these issues, this study develops surface-modified cartilage-targeting exosomes as non-viral carriers for gene therapy. Charge-reversed cationic exosomes are engineered for mRNA delivery by anchoring cartilage targeting optimally charged arginine-rich cationic motifs into the anionic exosome bilayer by using buffer pH as a charge-reversal switch. Cationic exosomes penetrated through the full-thickness of early-stage arthritic human cartilage owing to weak-reversible ionic binding with GAGs and efficiently delivered the encapsulated eGFP mRNA to chondrocytes residing in tissue deep layers, while unmodified anionic exosomes do not. When intra-articularly injected into destabilized medial meniscus mice knees with early-stage OA, mRNA loaded charge-reversed exosomes overcame joint clearance and rapidly penetrated into cartilage, creating an intra-tissue depot and efficiently expressing eGFP; native exosomes remained unsuccessful. Cationic exosomes thus hold strong translational potential as a platform technology for cartilage-targeted non-viral delivery of any relevant mRNA targets for OA treatment.

RESUMEN

Longitudinal bone growth, achieved through endochondral ossification, is accomplished by a cartilaginous structure, the physis or growth plate, comprised of morphologically distinct zones related to chondrocyte function: resting, proliferating and hypertrophic zones. The resting zone is a stem cell-rich region that gives rise to the growth plate, and exhibits regenerative capabilities in response to injury. We discovered a FoxA2+group of long-term skeletal stem cells, situated at the top of resting zone, adjacent the secondary ossification center, distinct from the previously characterized PTHrP+ stem cells. Compared to PTHrP+ cells, FoxA2+ cells exhibit higher clonogenicity and longevity. FoxA2+ cells exhibit dual osteo-chondro-progenitor activity during early postnatal development (P0-P28) and chondrogenic potential beyond P28. When the growth plate is injured, FoxA2+ cells expand in response to trauma, and produce physeal cartilage for growth plate tissue regeneration.

Asunto(s)

Placa de Crecimiento , Proteína Relacionada con la Hormona Paratiroidea , Cartílago , Condrocitos , Factor Nuclear 3-beta del Hepatocito/metabolismo , Células Madre

RESUMEN

We previously found that FoxA factors are necessary for chondrocyte differentiation. To investigate whether FoxA factors alone are sufficient to drive chondrocyte hypertrophy, we build a FoxA2 transgenic mouse in which FoxA2 cDNA is driven by a reiterated Tetracycline Response Element (TRE) and a minimal CMV promoter. This transgenic line was crossed with a col2CRE;Rosa26rtTA/+ mouse line to generate col2CRE;Rosa26rtTA/+;TgFoxA2+/- mice for inducible expression of FoxA2 in cartilage using doxycycline treatment. Ectopic expression of FoxA2 in the developing skeleton reveals skeletal defects and shorter skeletal elements in E17.5 mice. The chondro-osseous border was frequently mis-shaped in mutant mice, with small islands of col.10+ hypertrophic cells extending in the metaphyseal bone. Even though overexpression of FoxA2 causes an accumulation of hypertrophic chondrocytes, it did not trigger ectopic hypertrophy in the immature chondrocytes. This suggests that FoxA2 may need transcriptional co-factors (such as Runx2), whose expression is restricted to the hypertrophic zone, and absent in the immature chondrocytes. To investigate a potential FoxA2/Runx2 interaction in immature chondrocytes versus hypertrophic cells, we separated these two subpopulations by FACS to obtain CD24+CD200+ hypertrophic chondrocytes and CD24+CD200- immature chondrocytes and we ectopically expressed FoxA2 alone or in combination with Runx2 via lentiviral gene delivery. In CD24+CD200+ hypertrophic chondrocytes, FoxA2 enhanced the expression of chondrocyte hypertrophic markers collagen 10, MMP13, and alkaline phosphatase. In contrast, in the CD24+CD200- immature chondrocytes, neither FoxA2 nor Runx2 overexpression could induce ectopic expression of hypertrophic markers MMP13, alkaline phosphatase, or PTH/PTHrP receptor. Overall these findings mirror our in vivo data, and suggest that induction of chondrocyte hypertrophy by FoxA2 may require other factors in addition to Runx2 (i.e., Hif2α, MEF2C, or perhaps unknown factors), whose expression/activity is rate-limiting in immature chondrocytes.

Asunto(s)

Condrocitos , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Fosfatasa Alcalina/metabolismo , Animales , Huesos/metabolismo , Cartílago/metabolismo , Diferenciación Celular/genética , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Hipertrofia , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , Factores de Transcripción/metabolismo

RESUMEN

Vertebrate lonesome kinase (Vlk) is a secreted tyrosine kinase important for normal skeletogenesis during embryonic development. Vlk null mice (Vlk-/- ) are born with severe craniofacial and limb skeletal defects and die shortly after birth. We used a conditional deletion model to remove Vlk in limb bud mesenchyme (Vlk-Prx1 cKO) to assess the specific requirement for Vlk expression by skeletal progenitor cells during endochondral ossification, and an inducible global deletion model (Vlk-Ubq iKO) to address the role of Vlk during fracture repair. Deletion of Vlk with Prx1-Cre recapitulated the limb skeletal phenotype of the Vlk-/- mice and enabled us to study the postnatal skeleton as Vlk-Prx1 cKO mice survived to adulthood. In Vlk-Prx1 cKO adult mice, limbs remained shorter with decreased trabecular and cortical bone volumes. Both Vlk-Prx1 cKO and Vlk-Ubq iKO mice had a delayed fracture repair response but eventually formed bridging calluses. Furthermore, levels of phosphorylated osteopontin (OPN) were decreased in tibias of Vlk-Ubq iKO, establishing OPN as a Vlk substrate in bone. In summary, our data indicate that Vlk produced by skeletal progenitor cells influences the timing and extent of chondrogenesis during endochondral bone formation and fracture repair. © 2022 American Society for Bone and Mineral Research (ASBMR).

Asunto(s)

Condrogénesis , Osteogénesis , Animales , Huesos , Condrogénesis/genética , Extremidades , Ratones , Ratones Noqueados , Osteogénesis/genética , Proteínas Tirosina Quinasas

RESUMEN

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate lonesome kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet α-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets exhibit a significant decrease in several tyrosine phosphobands. Results of functional testing of VLK-deficient platelets show decreased protease-activated receptor 4-mediated and collagen-mediated platelet aggregation but normal responses to adenosine 5'-diphosphate. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased protease-activated receptor 4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets display strongly reduced platelet accumulation and fibrin formation after laser-induced injury of cremaster arterioles compared with control mice but with normal bleeding times. These studies show that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

Asunto(s)

Plaquetas/metabolismo , Activación Plaquetaria , Proteínas Tirosina Quinasas/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/patología , Eliminación de Gen , Células HEK293 , Humanos , Ratones Transgénicos , Proteínas Tirosina Quinasas/genética , Trombosis/patología

RESUMEN

: The coordinated development and function of bone-forming (osteoblasts) and bone-resorbing (osteoclasts) cells is critical for the maintenance of skeletal integrity and calcium homeostasis. An enhanced adipogenic versus osteogenic potential of bone marrow mesenchymal stem cells (MSCs) has been linked to bone loss associated with diseases such as diabetes mellitus, as well as aging and postmenopause. In addition to an inherent decrease in bone formation due to reduced osteoblast numbers, recent experimental evidence indicates that an increase in bone marrow adipocytes contributes to a disproportionate increase in osteoclast formation. Therefore, a potential strategy for therapeutic intervention in chronic bone loss disorders such as osteoporosis is to interfere with the pro-osteoclastogenic influence of marrow adipocytes. However, application of this approach is limited by the extremely complex regulatory processes in the osteoclastogenic program. For example, key regulators of osteoclastogenesis such as the receptor activator of nuclear factor-kappaB ligand (RANKL) and the soluble decoy receptor osteoprotegerin (OPG) are not only secreted by both osteoblasts and adipocytes, but are also regulated through several cytokines produced by these cell types. In this context, biologically active signaling molecules secreted from bone marrow adipocytes, such as chemerin, adiponectin, leptin, visfatin and resistin, can have a profound influence on the osteoclast differentiation program of hematopoietic stem cells (HSCs), and thus, hold therapeutic potential under disease conditions. In addition to these paracrine signals, adipogenic transcription factors including CCAAT/enhancer binding protein alpha (C/EBPα), C/EBP beta (C/EBPß) and peroxisome proliferator-associated receptor gamma (PPARγ) are also expressed by osteoclastogenic cells. However, in contrast to MSCs, activation of these adipogenic transcription factors in HSCs promotes the differentiation of osteoclast precursors into mature osteoclasts. Herein, we discuss the molecular mechanisms that link adipogenic signaling molecules and transcription factors to the osteoclast differentiation program and highlight therapeutic strategies targeting these mechanisms for promoting bone homeostasis.

Asunto(s)

Adipocitos/citología , Comunicación Celular , Diferenciación Celular , Osteoclastos/citología , Adipocitos/metabolismo , Animales , Humanos , Osteoclastos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Bone morphogenetic proteins (BMPs) are key regulators of skeletal development, growth, and repair. Although BMP signaling is required for synovial joint formation and is also involved in preserving joint function after birth, the role of specific BMP ligands in adult joint homeostasis remains unclear. The purpose of this study was to define the role of Bmp2 in the morphogenesis and maintenance of the knee joint. To do this, we first created Bmp2-LacZ and Gdf5-LacZ knock-in mice and compared their expression patterns in the developing and postnatal murine knee joint. We then generated a knockout mouse model using the Gdf5-cre transgene to specifically delete Bmp2 within synovial joint-forming cells. Joint formation, maturation, and homeostasis were analyzed using histology, immunohistochemistry, qRT-PCR, and atomic force microscopy (AFM)-based nanoindentation to assess the cellular, molecular, and biomechanical changes in meniscus and articular cartilage. Bmp2 is expressed in the articular cartilage and meniscus of the embryonic and adult mouse knee in a pattern distinct from Gdf5. The knee joints of the Bmp2 knockout mice form normally but fail to mature properly. In the absence of Bmp2, the extracellular matrix and shape of the meniscus are altered, resulting in functional deficits in the meniscus and articular cartilage that lead to a progressive osteoarthritis (OA) like knee pathology as the animals age. These findings demonstrate that BMP activity provided by Bmp2 is required for the maturation and maintenance of the murine knee joint and reveal a unique role for Bmp2 that is distinct from Gdf5 in knee joint biology. © 2018 American Society for Bone and Mineral Research.

Asunto(s)

Proteína Morfogenética Ósea 2/metabolismo , Extremidades/crecimiento & desarrollo , Articulaciones/crecimiento & desarrollo , Envejecimiento/patología , Animales , Fenómenos Biomecánicos , Cartílago Articular/metabolismo , Extremidades/embriología , Genes Reporteros , Factor 5 de Diferenciación de Crecimiento/metabolismo , Integrasas/metabolismo , Articulaciones/embriología , Ratones Noqueados , Osteoartritis/patología , Fenotipo

RESUMEN

OBJECTIVE: To generate knockin mice that express a tamoxifen-inducible Cre recombinase from the Prg4 locus (Prg4(GFPCreERt2) mice) and to use these animals to fate-map the progeny of Prg4-positive articular cartilage cells at various ages. METHODS: We crossed Prg4(GFPCreERt2) mice with Rosa26(floxlacZ) or Rosa26(mTmG) reporter strains, admin-istered tamoxifen to the double heterozygous offspring at different ages, and assayed Cre-mediated recom-bination by histochemistry and/or fluorescence microscopy. RESULTS: In 1-month-old mice, the expression of the Prg4(GFPCreERt2) allele mirrored the expression of endogenous Prg4 and, when tamoxifen was admin-istered for 10 days, caused Cre-mediated recombination in ∼70% of the superficial-most chondrocytes. Prg4(GFPCreERt2)-expressing cells were mostly confined to the top 3 cell layers of the articular cartilage in 1-month-old mice, but descendants of these cells were located in deeper regions of the articular cartilage in aged mice. On embryonic day 17.5, Prg4(GFPCreERt2)-expressing cells were largely restricted to the superficial-most cell layer of the forming joint, yet at ∼1 year, the progeny of these cells spanned the depth of the articular cartilage. CONCLUSION: Our results suggest that Prg4-expressing cells located at the joint surface in the embryo serve as a progenitor population for all deeper layers of the mature articular cartilage. Also, our findings indicate that Prg4(GFPCreERt2) is expressed by superficial chondrocytes in young mice, but expands into deeper regions of the articular cartilage as the animals age. The Prg4(GFPCreERt2) allele should be a useful tool for inducing efficient Cre-mediated recombination of loxP-flanked alleles at sites of Prg4 expression.

Asunto(s)

Cartílago Articular/metabolismo , Condrocitos/metabolismo , Proteoglicanos/metabolismo , Células Madre/metabolismo , Animales , Cartílago Articular/citología , Condrocitos/citología , Técnicas de Sustitución del Gen , Integrasas , Ratones , Proteoglicanos/genética , Células Madre/citología

RESUMEN

The relative timing of SHH and BMP signals controls whether presomitic mesoderm (PSM) cells will adopt either a chondrogenic or lateral plate mesoderm fate. Here we document that SHH-mediated induction of Nkx3.2 maintains the competence of somitic cells to initiate chondrogenesis in response to subsequent BMP signals by repressing BMP-dependent induction of GATA genes. Conversely, administration of BMP signals to PSM or forced expression of GATA family members in chick PSM explants blocks induction of hedgehog-dependent gene expression. We demonstrate that GATA factors can interact with Gli factors and can recruit the transcriptional co-factor FOG1 (ZFPM1) to the regulatory region of the mouse Gli1 gene, repressing the induction of Gli1 by SHH by binding to both GATA and Gli binding sites. Knockdown of FOG1 reverses the ability of GATA factors to repress Gli1 expression. Our findings uncover a novel role for GATA transcription factors as repressors of hedgehog signaling, and document that NKX3.2 maintains the ability of sclerotomal cells to express SHH transcriptional targets in the presence of BMP signals by repressing the induction of Gata4/5/6.

Asunto(s)

Proteínas Morfogenéticas Óseas/metabolismo , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA5/metabolismo , Factor de Transcripción GATA6/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Condrocitos/citología , Perfilación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Proteína con Dedos de Zinc GLI1

RESUMEN

In the limb bud, patterning along the anterior-posterior (A-P) axis is controlled by Sonic Hedgehog (Shh), a signaling molecule secreted by the "Zone of Polarizing Activity", an organizer tissue located in the posterior margin of the limb bud. We have found that the transcription factors GATA4 and GATA6, which are key regulators of cell identity, are expressed in an anterior to posterior gradient in the early limb bud, raising the possibility that GATA transcription factors may play an additional role in patterning this tissue. While both GATA4 and GATA6 are expressed in an A-P gradient in the forelimb buds, the hindlimb buds principally express GATA6 in an A-P gradient. Thus, to specifically examine the role of GATA6 in limb patterning we generated Prx1-Cre; GATA6(fl/fl) mice, which conditionally delete GATA6 from their developing limb buds. We found that these animals display ectopic expression of both Shh and its transcriptional targets specifically in the anterior mesenchyme of the hindlimb buds. Loss of GATA6 in the developing limbs results in the formation of preaxial polydactyly in the hindlimbs. Conversely, forced expression of GATA6 throughout the limb bud represses expression of Shh and results in hypomorphic limbs. We have found that GATA6 can bind to chromatin (isolated from limb buds) encoding either Shh or Gli1 regulatory elements that drive expression of these genes in this tissue, and demonstrated that GATA6 works synergistically with FOG co-factors to repress expression of luciferase reporters driven by these sequences. Most significantly, we have found that conditional loss of Shh in limb buds lacking GATA6 prevents development of hindlimb polydactyly in these compound mutant embryos, indicating that GATA6 expression in the anterior region of the limb bud blocks hindlimb polydactyly by repressing ectopic expression of Shh.

Asunto(s)

Tipificación del Cuerpo/genética , Factor de Transcripción GATA6/biosíntesis , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/metabolismo , Polidactilia/genética , Animales , Embrión de Mamíferos , Desarrollo Embrionario , Miembro Anterior/crecimiento & desarrollo , Miembro Anterior/metabolismo , Factor de Transcripción GATA4/biosíntesis , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/biosíntesis , Proteínas Hedgehog/genética , Miembro Posterior/crecimiento & desarrollo , Miembro Posterior/metabolismo , Ratones , Polidactilia/etiología , Polidactilia/patología , Transducción de Señal/genética

RESUMEN

During endochondral ossification, small, immature chondrocytes enlarge to form hypertrophic chondrocytes, which express collagen X. In this work, we demonstrate that FoxA factors are induced during chondrogenesis, bind to conserved binding sites in the collagen X enhancer, and can promote the expression of a collagen X-luciferase reporter in both chondrocytes and fibroblasts. In addition, we demonstrate by both gain- and loss-of-function analyses that FoxA factors play a crucial role in driving the expression of both endogenous collagen X and other hypertrophic chondrocyte-specific genes. Mice engineered to lack expression of both FoxA2 and FoxA3 in their chondrocytes display defects in chondrocyte hypertrophy, alkaline phosphatase expression, and mineralization in their sternebrae and, in addition, exhibit postnatal dwarfism that is coupled to significantly decreased expression of both collagen X and MMP13 in their growth plates. Our findings indicate that FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program.

Asunto(s)

Aumento de la Célula , Condrocitos/metabolismo , Condrogénesis/genética , Colágeno Tipo X/metabolismo , Enanismo/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-gamma del Hepatocito/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Sitios de Unión , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Condrocitos/citología , Colágeno Tipo X/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Enanismo/embriología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Genes Reporteros , Placa de Crecimiento/metabolismo , Factor Nuclear 3-beta del Hepatocito/deficiencia , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-gamma del Hepatocito/deficiencia , Factor Nuclear 3-gamma del Hepatocito/genética , Metaloproteinasa 13 de la Matriz/genética , Huesos Metatarsianos/citología , Huesos Metatarsianos/metabolismo , Ratones , Ratones Mutantes , Factores Reguladores Miogénicos/metabolismo , Proteína Smad1/metabolismo

RESUMEN

Whereas Runx2 is necessary for bone formation and cartilage hypertrophy, it is unclear why Runx2 induces markers of chondrocyte hypertrophy only in chondrocytes. We document that chondrocytes either contain a cofactor, which can be induced in somitic cells by prochondrogenic signals, that is necessary for Runx2 to induce chondrocyte hypertrophy or, alternatively, lack a repressor of this maturation program. Sequential Shh and bone morphogenetic protein (BMP) signals or forced expression of either Nkx3.2 or Sox9 (plus BMP signals) induces chondrogenesis in presomitic mesoderm and simultaneously induces a competence for Runx2 to activate the chondrocyte maturation program. The ability of either sequential Shh and BMP signals or retrovirus-encoded Nkx3.2 or Sox9 to induce this competence correlates with their ability to activate chondrogenesis in various embryonic tissues. Consistent with these findings in embryonic tissues, we have found that cotransfected Runx2 and Smad1 are able to induce the expression of a reporter construct driven by the collagen X regulatory sequences in chondrocytes but not in fibroblasts. In contrast, both Runx2 and Smad1 are competent to activate reporters driven by either reiterated Runx or Smad binding sites, respectively, in both cell types. As Sox9 and Nkx3.2 have previously been shown to block chondrocyte maturation in vivo, our findings suggest that these transcription factors can, in addition, either induce the expression or activity of a factor in chondrocytes that is required for Runx2 to activate the chondrocyte maturation program, or alternatively that these transcription factors block the expression or activity of a repressor of this maturation program.

Asunto(s)

Condrocitos/metabolismo , Condrogénesis/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Animales , Embrión de Pollo , Mesodermo/citología , Mesodermo/metabolismo , Transducción de Señal/fisiología

RESUMEN

Prostaglandins are ubiquitous metabolites of arachidonic acid, and cyclooxygenase inhibitors prevent their production and secretion. Animals with loss of cyclooxygenase-2 function have reduced reparative bone formation, but the role of prostaglandins during endochondral bone formation is not defined. The role of PGE2 as a regulator of chondrocyte differentiation in chick growth plate chondrocytes (GPCs) was examined. While PGE2, PGD2, PGF2alpha, and PGJ2 all inhibited colX expression, approximately 80% at 10(-6) M, PGE2 was the most potent activator of cAMP response element (CRE)-mediated transcription. PGE2 dose-dependently inhibited the expression of the differentiation-related genes, colX, VEGF, MMP-13, and alkaline phosphatase gene, and enzyme activity with significant effects at concentrations as low as 10(-10) M. PGE2 induced cyclic AMP response element binding protein (CREB) phosphorylation and increased c-Fos protein levels by 5 min, and activated transcription at CRE-Luc, AP-1-Luc, and c-Fos promoter constructs. The protein kinase A (PKA) inhibitor, H-89, completely blocked PGE2-mediated induction of CRE-Luc and c-Fos promoter-Luc promoters, and partially inhibited induction of AP-1-Luc, while the protein kinase C (PKC) inhibitor Go-6976 partially inhibited all three promoters, demonstrating substantial cross-talk between these signaling pathways. PGE2 inhibition of colX gene expression was dependent upon both PKA and PKC signaling. These observations demonstrate potent prostaglandin regulatory effects on chondrocyte maturation and show a role for both PKA and PKC signaling in PGE2 regulatory events.

Asunto(s)

Diferenciación Celular/fisiología , Condrocitos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/fisiología , Osteogénesis/fisiología , Proteína Quinasa C/metabolismo , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Cartílago/crecimiento & desarrollo , Cartílago/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Pollos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Colágeno Tipo X/efectos de los fármacos , Colágeno Tipo X/metabolismo , Colagenasas/efectos de los fármacos , Colagenasas/metabolismo , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Dinoprostona/metabolismo , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Metaloproteinasa 13 de la Matriz , Osteogénesis/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteína Quinasa C/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The bone-related transcription factor Runx2 (Cbfa1) has been extensively shown to regulate osteoblast differentiation and function. Recent studies demonstrate that Runx2 is also a positive regulator of chondrocyte maturation and vascular invasion in cartilage. Runx2 activity can be modulated in several ways, including direct stimulation of gene expression, post-translational modification, and protein-protein interactions. We have previously reported cooperative effects between BMP and RA downstream signaling involving Smad proteins and Runx2. Furthermore, our previous studies showed that PTHrP inhibits chondrocyte maturation primarily through CREB and AP-1 signaling pathways. In the present study, we investigated the effect of PTHrP on Runx2 expression in chick upper sternal chondrocytes (USCs). We further determined the signaling pathways through which PTHrP regulates Runx2 transcription. Our results show that PTHrP inhibits Runx2 expression at both the mRNA and protein levels concomitant with a PTHrP-mediated suppression of the phenotypic marker of hypertrophy, type X collagen. We further determined potential signaling pathways through which PTHrP inhibits Runx2 expression using protein kinase inhibitors, H89 (PKA inhibitor): Go-6976 (PKC inhibitor): SB203850 (p38 MAPK inhibitor), and U0126 (MEK inhibitor). We show that pretreatment with PKA and, to a lesser extent, PKC inhibitors significantly blocked PTHrP suppression of Runx2, while p38 MAPK and MEK inhibitors had no significant effect. Furthermore, PTHrP suppression of Runx2 mRNA was partially blocked in USCs infected with RCAS-A-CREB, a dominant negative reagent that abrogates CREB activity. Overall, our results demonstrate that PTHrP downregulates Runx2 expression primarily through the PKA signaling pathway.

Asunto(s)

Cartílago/enzimología , Cartílago/crecimiento & desarrollo , Condrocitos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Neoplasias/metabolismo , Osteogénesis/fisiología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Colágeno Tipo X/efectos de los fármacos , Colágeno Tipo X/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/antagonistas & inhibidores

RESUMEN

Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF-2 and AP-1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP-2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP-2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP-2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP-2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter-specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes.

Asunto(s)

Proteínas Morfogenéticas Óseas/farmacología , Condrocitos/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas de Unión al ADN/genética , Transactivadores/genética , Activación Transcripcional/fisiología , Factor de Crecimiento Transformador beta , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2 , Diferenciación Celular/fisiología , Células Cultivadas , Colágeno Tipo X/efectos de los fármacos , Colágeno Tipo X/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico , Proteínas de Unión al ADN/efectos de los fármacos , Ensayo de Cambio de Movilidad Electroforética , Proteínas Hedgehog , Datos de Secuencia Molecular , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Regiones Promotoras Genéticas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína smad6 , Transactivadores/efectos de los fármacos , Transactivadores/fisiología , Transcripción Genética/fisiología

RESUMEN

BMPs regulate cartilage differentiation and have been approved for clinical use as stimulators of bone repair. BMP signaling is complex and there are multiple potential points of regulation, including modulation of Smad signaling, which is inhibited by both Smad6 and Smad7. In the current manuscript we assessed the expression and biological function of Smad6 during chondrocyte differentiation. We found that the induction of chondrocyte differentiation by BMP-2 in chicken sternal embryonic chondrocytes was accompanied by a marked increase in Smad6 mRNA and protein levels. A morpholino antisense oligonucleotide complementary to Smad6 reduced the expression of Smad6 protein and enhanced the stimulatory effect of BMP-2 on both colX and alkaline phosphatase activity. In contrast, over-expression of Smad6 blocked BMP-2 mediated induction of the type X collagen promoter, b2-640 Luc. Therefore, expression studies as well as gain and loss of function experiments suggest that Smad6 participates in an important negative feedback loop whereby BMP-2 mediated effects on chondrocyte differentiation are reduced by induction of Smad6. Additional studies are required to determine the extent to which this pathway participates in pathologic processes involving cartilage.

Asunto(s)

Proteínas Morfogenéticas Óseas/fisiología , Condrocitos/citología , Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Embrión de Pollo , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/fisiología , Proteínas de Unión al ADN/metabolismo , Proteína smad6 , Transactivadores/metabolismo

RESUMEN

This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-beta. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-beta, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-beta-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-beta. However, in combination the effects were additive and similar to the inhibitory effects of TGF-beta on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-beta on colX, while together the blockade was complete. Similar effects were observed with another TGF-beta-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-beta signaling. In contrast, although TGF-beta does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-beta and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-beta signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-beta or BMP-2.

Asunto(s)

Condrocitos/citología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta/farmacología , Factor de Transcripción Activador 2 , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Embrión de Pollo , Condrocitos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptor Cross-Talk , Transducción de Señal , Proteína smad3 , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Whereas bone morphogenetic protein (BMP)-signaling events induce maturational characteristics in vitro, recent evidence suggests that the effects of other regulators might be mediated through BMP-signaling events. The present study examines the mechanism through which retinoic acid (RA) stimulates differentiation in chicken embryonic caudal sternal chondrocyte cultures. Both RA and BMP-2 induced expression of the chondrocyte maturational marker, colX, in chondrocyte cultures by 8 d. Though the RA effect was small, it synergistically enhanced the effect of BMP-2 on colX and phosphatase activity. Inhibition of either RA or BMP signaling, with selective inhibitors, interfered with the inductive effects of these agents but also inhibited the complementary pathway, demonstrating a codependence of RA and BMP signaling during chondrocyte maturation. BMP-2 did not enhance the effects of RA on an RA-responsive reporter construct, but RA enhanced basal activity and synergistically enhanced BMP-2 stimulation of the BMP-responsive chicken type X collagen reporter. A similar synergistic interaction between RA and BMP-2 was observed on colX expression. RA did not increase the expression of the type IA BMP receptor but did markedly up-regulate the expression of Smad1 and Smad5 proteins, important participants in the BMP pathway. Inhibition of RA signaling, with the selective inhibitor AGN 193109, blocked RA-mediated induction of the Smad proteins and chondrocyte differentiation. These findings demonstrate that RA induces the expression of BMP-signaling molecules and enhances BMP effects in chondrocytes.

Asunto(s)

Antineoplásicos/farmacología , Proteínas Morfogenéticas Óseas/farmacología , Condrocitos/citología , Proteínas de Unión al ADN/genética , Transactivadores/genética , Factor de Crecimiento Transformador beta , Tretinoina/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Diferenciación Celular/efectos de los fármacos , Embrión de Pollo , Pollos , Condrocitos/fisiología , Colágeno Tipo X/genética , Sinergismo Farmacológico , Fosfoproteínas/genética , Transducción de Señal/efectos de los fármacos , Proteínas Smad , Proteína Smad5 , Esternón/citología

RESUMEN

Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca(2+) concentration ([Ca(2+)](i)) from 50 to 100 nM. To determine if this rise in [Ca(2+)](i) functionally participates in the maturational process of growth plate chondrocytes (GPCs), we examined its effect on several markers of hypertrophy, including annexin V, bone morphogenetic protein-6, type X collagen, and indian hedgehog. Expression of these genes was determined under conditions either where the Ca(2+) chelator EGTA was used to deplete extracellular Ca(2+) and lower [Ca(2+)](i) to < 50 nM or where the extracellular addition of 5 mM CaCl(2) was used to elevate [Ca(2+)](i) to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl(2), 4 mM EGTA significantly inhibited their expression. This effect was recapitulated in sternal chondrocytes and was reversed following withdrawal of EGTA. Based on these findings, we hypothesized that the EGTA-induced suppression of these genes was mediated by a factor whose expression is responsive to changes in basal [Ca(2+)](i). Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca(2+)](i) on PTHrP expression. Suggesting that low [Ca(2+)](i) suppression of hypertrophy was PTHrP-dependent in GPCs, (a) treatment with 4 mM EGTA increased PTHrP expression, (b) the EGTA effect was rescued by blocking PTHrP binding to its receptor with the competitive antagonist TIP(7-39), and (c) EGTA could mimic the PTHrP stimulation of AP-1 binding to DNA. Additionally, PTHrP promoter analysis identified a domain (-1498 to -862, relative to the start codon) involved with conferring Ca(2+) sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca(2+) in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca(2+)](i).

Asunto(s)

Señalización del Calcio/genética , Calcio/deficiencia , Diferenciación Celular/genética , Condrocitos/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Placa de Crecimiento/embriología , Líquido Intracelular/metabolismo , Osteogénesis/genética , Animales , Animales Recién Nacidos , Anexina A5/genética , Anexina A5/metabolismo , Proteína Morfogenética Ósea 6 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quelantes , Embrión de Pollo , Pollos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Placa de Crecimiento/crecimiento & desarrollo , Placa de Crecimiento/metabolismo , Proteínas Hedgehog , Hipertrofia/genética , Hipertrofia/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética