RESUMEN
Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.
Asunto(s)
Calcio , Hipofosfatemia , Ratones , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Osteoblastos/metabolismo , Osteocitos/metabolismo , Fosfatos/metabolismoRESUMEN
In response to mechanical loading of bone, osteocytes produce nitric oxide (NOâ¢) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO⢠production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO⢠as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO⢠donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO⢠production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NOâ¢, and that NO⢠is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO⢠production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO⢠production following FSS, indicating that CaMKII is needed for NO⢠production. However, NO⢠donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO⢠may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NOâ¢, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Óxido Nítrico , Osteocitos , Óxido Nítrico/metabolismo , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Osteocitos/metabolismo , Ratones , Estrés Mecánico , Ratones Endogámicos C57BL , Mecanotransducción Celular , Línea Celular , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismoRESUMEN
Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.
Asunto(s)
Mama/patología , Calcio/metabolismo , Mecanotransducción Celular , NADPH Oxidasa 2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Catiónicos TRPM/metabolismo , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Microtúbulos/metabolismo , NADPH Oxidasa 2/genética , Pronóstico , Proteínas Proto-Oncogénicas p21(ras)/genética , Tasa de Supervivencia , Canales Catiónicos TRPM/genética , Microambiente TumoralRESUMEN
Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of α-motor neurons, leading to profound skeletal muscle atrophy. Patients also suffer from decreased bone mineral density and increased fracture risk. The majority of treatments for SMA, approved or in clinic trials, focus on addressing the underlying cause of disease, insufficient production of full-length SMN protein. While restoration of SMN has resulted in improvements in functional measures, significant deficits remain in both mice and SMA patients following treatment. Motor function in SMA patients may be additionally improved by targeting skeletal muscle to reduce atrophy and improve muscle strength. Inhibition of myostatin, a negative regulator of muscle mass, offers a promising approach to increase muscle function in SMA patients. Here we demonstrate that muSRK-015P, a monoclonal antibody which specifically inhibits myostatin activation, effectively increases muscle mass and function in two variants of the pharmacological mouse model of SMA in which pharmacologic restoration of SMN has taken place either 1 or 24 days after birth to reflect early or later therapeutic intervention. Additionally, muSRK-015P treatment improves the cortical and trabecular bone phenotypes in these mice. These data indicate that preventing myostatin activation has therapeutic potential in addressing muscle and bone deficiencies in SMA patients. An optimized variant of SRK-015P, SRK-015, is currently in clinical development for treatment of SMA.
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Atrofia Muscular Espinal/genética , Miostatina/genética , Miostatina/fisiología , Animales , Anticuerpos Monoclonales , Modelos Animales de Enfermedad , Ratones , Neuronas Motoras/metabolismo , Fuerza Muscular/fisiología , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/fisiopatología , Miostatina/antagonistas & inhibidores , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin (mdx), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Distrofina/deficiencia , Mecanotransducción Celular , Contracción Muscular , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Modelos Animales de Enfermedad , Distrofina/genética , Ratones Endogámicos mdx , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatología , Fosforilación , Proteínas Señalizadoras YAPRESUMEN
Connexin43 is an important modulator of many signaling pathways in bone. ß-Catenin, a key regulator of the osteoblast differentiation and function, is among the pathways downstream of connexin43-dependent intercellular communication. There are striking overlaps between the functions of these two proteins in bone cells. However, differential effects of connexin43 on ß-catenin activity have been reported. Here, we examined how connexin43 influenced both Wnt-dependent and Wnt-independent activation of ß-catenin in osteoblasts in vitro. Our data show that loss of connexin43 in primary osteoblasts or connexin43 overexpression in UMR106 cells regulated active ß-catenin and phospho-Akt levels, with loss of connexin43 inhibiting and connexin43 overexpression increasing the levels of active ß-catenin and phospho-Akt. Increasing connexin43 expression synergistically enhanced Wnt3a-dependent activation of ß-catenin protein and ß-catenin transcriptional activity, as well as Wnt-independent activation of ß-catenin by prostaglandin E2 (PGE2). Finally, we show that the activation of ß-catenin by PGE2 required signaling through the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3ß) pathway, as the PI3K inhibitor, LY-294002, disrupted the synergy between connexin43 and PGE2. These data show that connexin43 regulates Akt and ß-catenin activity and synergistically enhances both Wnt-dependent and Wnt-independent ß-catenin signaling in osteoblasts.
Asunto(s)
Conexina 43/metabolismo , Dinoprostona/metabolismo , Osteoblastos/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal/fisiología , Transcripción Genética/fisiologíaRESUMEN
In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/- controls, male Gja1-/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and ß-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition.
Asunto(s)
Remodelación Ósea , Conexina 43/química , Conexina 43/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Transducción de Señal , Animales , Resorción Ósea/patología , Calcificación Fisiológica , Diferenciación Celular , Proliferación Celular , Colágeno/metabolismo , Hueso Cortical/metabolismo , Matriz Extracelular/metabolismo , Masculino , Ratones , Modelos Animales , Fenotipo , Porosidad , Unión Proteica , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In bone, connexin43 expression in cells of the osteoblast lineage plays an important role in restraining osteoclastogenesis and bone resorption. While there is a consensus around the notion that the anti-osteoclastogenic factor, osteoprotegerin, is a driver of this effect, how connexin43 regulates osteoprotegerin gene expression is unclear. Here, we show that loss of connexin43 decreased osteoprotegerin gene expression and reduced ERK1/2 activation. Conversely, overexpression of connexin43 increased osteoprotegerin expression and enhanced ERK1/2 activation. This increase in phospho-ERK1/2 is required for connexin43 to induce transcription from the osteoprotegerin proximal promoter. Connexin43 increased promoter activity via a specific 200 base pair region of the osteoprotegerin promoter located at -1486 to -1286 with respect to the transcriptional start site, a region which includes four Sp1 binding elements. Further, activation of this promoter region required an intact functional connexin43, as hypomorphic or dominant negative connexin43 mutant constructs, including one with increased hemichannel activity, were unable to stimulate osteoprotegerin expression as strongly as wild type connexin43. Using chromatin immunoprecipitations, we show that connexin43 expression enhanced the recruitment of Sp1, but not Runx2, to the osteoprotegerin proximal promoter. In total, these data show that connexin43-dependent gap junctional communication among osteoblast cells permits efficient ERK1/2 activation. ERK1/2 signaling promotes the recruitment of the potent transcriptional activator, Sp1, to the osteoprotegerin proximal promoter, resulting in robust transcription of anti-osteoclastogenic factor, osteoprotegerin.
Asunto(s)
Conexina 43/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoprotegerina/genética , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Animales , Células Cultivadas , Ratones Endogámicos C57BL , Osteoblastos/citología , Osteoblastos/metabolismo , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: Although use of nonsteroidal antiinflammatory drugs and low-dose irradiation has demonstrated efficacy in preventing heterotopic ossification (HO) after THA and surgical treatment of acetabular fractures, these modalities have not been assessed after traumatic blast amputations where HO is a common complication that can arise in the residual limb. QUESTIONS/PURPOSES: The purpose of this study was to investigate the effectiveness of indomethacin and irradiation in preventing HO induced by high-energy blast trauma in a rat model. METHODS: Thirty-six Sprague-Dawley rats underwent hind limb blast amputation with a submerged explosive under water followed by irrigation and primary wound closure. One group (n = 12) received oral indomethacin for 10 days starting on postoperative Day 1. Another group (n = 12) received a single dose of 8 Gy irradiation to the residual limb on postoperative Day 3. A control group (n = 12) did not receive either. Wound healing and clinical course were monitored in all animals until euthanasia at 24 weeks. Serial radiographs were taken immediately postoperatively, at 10 days, and every 4 weeks thereafter to monitor the time course of ectopic bone formation until euthanasia. Five independent graders evaluated the 24-week radiographs to quantitatively assess severity and qualitatively assess the pattern of HO using a modified Potter scale from 0 to 3. Assessment of grading reproducibility yielded a Fleiss statistic of 0.41 and 0.37 for severity and type, respectively. By extrapolation from human clinical trials, a minimum clinically important difference in HO severity was empirically determined to be two full grades or progression of absolute grade to the most severe. RESULTS: We found no differences in mean HO severity scores among the three study groups (indomethacin 0.90 ± 0.46 [95% confidence interval {CI}, 0.60-1.19]; radiation 1.34 ± 0.59 [95% CI, 0.95-1.74]; control 0.95 ± 0.55 [95% CI, 0.60-1.30]; p = 0.100). For qualitative HO type scores, the radiation group had a higher HO type than both indomethacin and controls, but indomethacin was no different than controls (indomethacin 1.08 ± 0.66 [95% CI, 0.67-1.50]; radiation 1.89 ± 0.76 [95% CI, 1.38-2.40]; control 1.10 ± 0.62 [95% CI, 0.70-1.50]; p = 0.013). The lower bound of the 95% CI on mean severity in the indomethacin group and the upper bound of the radiation group barely spanned a full grade and involved only numeric grades < 2, suggesting that even if a small difference in severity could be detected, it would be less than our a priori-defined minimum clinically important difference and any differences that might be present are unlikely to be clinically meaningful. CONCLUSIONS: This work unexpectedly demonstrated that, compared with controls, indomethacin and irradiation provide no effective prophylaxis against HO in the residual limb after high-energy blast amputation in a rat model. Such an observation is contrary to the civilian experience and may be potentially explained by either a different pathogenesis for blast-induced HO or a stimulus that overwhelms conventional regimens used to prevent HO in the civilian population. CLINICAL RELEVANCE: HO in the residual limb after high-energy traumatic blast amputation will likely require novel approaches for prevention and management.
Asunto(s)
Amputación Traumática/terapia , Antiinflamatorios no Esteroideos/farmacología , Traumatismos por Explosión/terapia , Indometacina/farmacología , Osificación Heterotópica/prevención & control , Dosis de Radiación , Amputación Traumática/etiología , Animales , Traumatismos por Explosión/etiología , Modelos Animales de Enfermedad , Masculino , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/etiología , Ratas Sprague-Dawley , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
Shaping of the skeleton (modeling) and its maintenance throughout life (remodeling) require coordinated activity among bone forming (osteoblasts) and resorbing cells (osteoclasts) and osteocytes (bone embedded cells). The gap junction protein connexin43 (Cx43) has emerged as a key modulator of skeletal growth and homeostasis. The skeletal developmental abnormalities present in oculodentodigital and craniometaphyseal dysplasias, both linked to Cx43 gene (GJA1) mutations, demonstrate that the skeleton is a major site of Cx43 action. Via direct action on osteolineage cells, including altering production of pro-osteoclastogenic factors, Cx43 contributes to peak bone mass acquisition, cortical modeling of long bones, and maintenance of bone quality. Cx43 also contributes in diverse ways to bone responsiveness to hormonal and mechanical signals. Skeletal biology research has revealed the complexity of Cx43 function; in addition to forming gap junctions and "hemichannels", Cx43 provides a scaffold for signaling molecules. Hence, Cx43 actively participates in generation and modulation of cellular signals driving skeletal development and homeostasis. Pharmacological interference with Cx43 may in the future help remedy deterioration of bone quality occurring with aging, disuse and hormonal imbalances.
Asunto(s)
Huesos/metabolismo , Conexinas/metabolismo , Animales , Desarrollo Óseo , Enfermedades Óseas/metabolismo , Conexinas/química , Homeostasis , Humanos , Investigación Biomédica TraslacionalRESUMEN
PURPOSE OF THE REVIEW: This review highlights recent developments into how intercellular communication through connexin43 facilitates bone modeling and remodeling. RECENT FINDINGS: Connexin43 is required for both skeletal development and maintenance, particularly in cortical bone, where it carries out multiple functions, including preventing osteoclastogenesis, restraining osteoprogenitor proliferation, promoting osteoblast differentiation, coordinating organized collagen matrix deposition, and maintaining osteocyte survival. Emerging data shows that connexin43 regulates both the exchange of small molecules among osteoblast lineage cells and the docking of signaling proteins to the gap junction, affecting the efficiency of signal transduction. Understanding how and what connexin43 communicates to coordinate tissue remodeling has therapeutic implications in bone. Altering the information shared by intercellular communication and/or targeting the recruitment of signaling machinery to the gap junction could be used to impact the skeletal homeostatic set point, either driving osteogenesis or inhibiting resorption.
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Desarrollo Óseo , Remodelación Ósea , Huesos/metabolismo , Conexina 43/metabolismo , Animales , Comunicación Celular , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Colágeno/metabolismo , Hueso Cortical/metabolismo , Matriz Extracelular/metabolismo , Uniones Comunicantes/metabolismo , Humanos , Osteoblastos , Osteocitos , Osteogénesis , Células MadreRESUMEN
While the type IV intermediate filament protein, synemin, has been shown to play a role in striated muscle and neuronal tissue, its presence and function have not been described in skeletal tissue. Here, we report that genetic ablation of synemin in 14-wk-old male mice results in osteopenia that includes a more than 2-fold reduction in the trabecular bone fraction in the distal femur and a reduction in the cross-sectional area at the femoral middiaphysis due to an attendant reduction in both the periosteal and endosteal perimeter. Analysis of serum markers of bone formation and static histomorphometry revealed a statistically significant defect in osteoblast activity and osteoblast number in vivo. Interestingly, primary osteoblasts isolated from synemin-null mice demonstrate markedly enhanced osteogenic capacity with a concomitant reduction in cyclin D1 mRNA expression, which may explain the loss of osteoblast number observed in vivo. In total, these data suggest an important, previously unknown role for synemin in bone physiology.
Asunto(s)
Densidad Ósea/fisiología , Hueso Esponjoso/metabolismo , Fémur/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/metabolismo , Osteogénesis/fisiología , Animales , Biomarcadores/sangre , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/fisiopatología , Hueso Esponjoso/fisiología , Diferenciación Celular/fisiología , Ciclina D1/metabolismo , Fémur/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Osteoblastos/metabolismo , Osteoblastos/fisiología , ARN Mensajero/metabolismoRESUMEN
Regulation of bone homeostasis depends on the concerted actions of bone-forming osteoblasts and bone-resorbing osteoclasts, controlled by osteocytes, cells derived from osteoblasts surrounded by bone matrix. The control of differentiation, viability and function of bone cells relies on the presence of connexins. Connexin43 regulates the expression of genes required for osteoblast and osteoclast differentiation directly or by changing the levels of osteocytic genes, and connexin45 may oppose connexin43 actions in osteoblastic cells. Connexin37 is required for osteoclast differentiation and its deletion results in increased bone mass. Less is known on the role of connexins in cartilage, ligaments and tendons. Connexin43, connexin45, connexin32, connexin46 and connexin29 are expressed in chondrocytes, while connexin43 and connexin32 are expressed in ligaments and tendons. Similarly, although the expression of pannexin1, pannexin2 and pannexin3 has been demonstrated in bone and cartilage cells, their function in these tissues is not fully understood.
Asunto(s)
Huesos/metabolismo , Huesos/fisiología , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Uniones Comunicantes/fisiología , Canales Iónicos/metabolismo , Animales , Homeostasis/fisiología , HumanosRESUMEN
Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated significant alterations in the neuromuscular junction (NMJ) morphology and resulting neuromuscular transmission failure (NTF) 24 h after injury in mdx mice (murine model for DMD). Here we determine the contribution of NMJ morphology and NTF to the recovery of muscle contractile function post-injury. NMJ morphology and NTF rates were assessed day 0 (immediately after injury) and days 1, 7, 14 and 21 after quadriceps injury. Eccentric injury of the quadriceps resulted in a significant loss of maximal torque in both WT (39 ± 6 %) and mdx (76 ± 8 %) with a full recovery in WT by day 7 and in mdx by day 21. Post-injury alterations in NMJ morphology and NTF were found only in mdx, were limited to days 0 and 1, and were independent of changes in MuSK or AChR expression. Such early changes at the NMJ after injury are consistent with mechanical disruption rather than newly forming NMJs. Furthermore, we show that the dense microtubule network that underlies the NMJ is significantly reduced and disorganized in mdx compared to WT. These structural changes at the NMJ may play a role in the increased NMJ disruption and the exaggerated loss of nerve-evoked muscle force seen after injury to dystrophic muscles.
Asunto(s)
Distrofina/fisiología , Distrofia Muscular de Duchenne/patología , Unión Neuromuscular/lesiones , Unión Neuromuscular/metabolismo , Regeneración/fisiología , Animales , Western Blotting , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Contracción Muscular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Unión Neuromuscular/fisiopatología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Colinérgicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The biologic factors associated with shoulder osteoarthritis (OA) have not been elucidated. The purpose of this study was to investigate osteoarthritic biomarkers of the shoulder. To our knowledge, this is the first study to analyze shoulder cartilage for OA-associated genes and to examine human shoulder cartilage for a possible biomarker, connexin 43 (Cx43). MATERIALS AND METHODS: Cartilage from 16 osteoarthritic and 10 nonosteoarthritic humeral heads was assessed for expression of the following genes by real-time polymerase chain reaction: types I, II, and X collagen; matrix metalloproteinases (MMPs); tissue inhibitors of MMP (TIMPs); interleukins; versican; cyclooxygenase 2 (Cox-2); inducible nitric oxide synthase (iNOS); tumor necrosis factor α (TNF-α); aggrecanase 2 (ADAMTS5); and Cx43. RESULTS: In osteoarthritic shoulders, Cx43, Cox-2, versican, collagen type I, ADAMTS5, MMP-3, and TNF-α expressions were significantly increased compared with controls. TIMP-3 and iNOS trended toward significance, with robust expression in osteoarthritic shoulders and low expression in nonosteoarthritic shoulders. In osteoarthritic shoulders, gene expression of Cx43, ADAMTS5, collagen type I, Cox-2, versican, and TIMP-3 showed predominance (85-, 33-, 13-, 12-, 11.5-, and 3-fold increases, respectively) relative to nonosteoarthritic controls. Spearman correlation analysis showed significant correlations between Cx43 and collagen (types I, II, and X), MMP-9, TIMP-2 and TIMP-3, versican, Cox-2, iNOS, and ADAMTS5. CONCLUSIONS: Certain genes are markedly upregulated in osteoarthritic shoulders compared with nonosteoarthritic shoulders, with Cx43, Cox-2, versican, collagen type I, ADAMTS5, MMP-3, and TNF-α expression being significantly increased. These genes might be useful biomarkers for examining shoulder OA. CLINICAL RELEVANCE: Identification of osteoarthritic biomarkers can help us better understand shoulder OA and build the foundation for future research on disease progression and treatments.
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Biomarcadores/metabolismo , Cartílago Articular/química , Conexina 43/análisis , Cabeza Humeral/química , Osteoartritis/metabolismo , Articulación del Hombro/química , Proteínas ADAM/biosíntesis , Proteína ADAMTS5 , Adulto , Anciano , Colágeno/biosíntesis , Ciclooxigenasa 2/biosíntesis , Femenino , Humanos , Interleucinas/biosíntesis , Masculino , Metaloproteinasas de la Matriz/biosíntesis , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Osteoartritis/genética , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba , Versicanos/biosíntesisRESUMEN
Osteoblasts, osteocytes, and osteoprogenitor cells are interconnected into a functional network by gap junctions formed primarily by connexin43 (Cx43). Over the past two decades, it has become clear that Cx43 is important for the function of osteoblasts and osteocytes. This connexin contributes to the acquisition of peak bone mass and is a major modulator of cortical modeling. We review key data from human and mouse genetics on the skeletal consequences of ablation or mutation of the Cx43 gene (Gja1) and the molecular mechanisms by which Cx43 regulates the differentiation, function, and survival of osteogenic lineage cells. We also discuss putative second messengers that are communicated by Cx43 gap junctions, the role of hemichannels, and the function of Cx43 as a scaffold for signaling molecules. Current knowledge demonstrates that Cx43 is more than a passive channel; rather, it actively participates in the generation and modulation of cellular signals that drive skeletal development and homeostasis.
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Diferenciación Celular/fisiología , Conexina 43/metabolismo , Osteoblastos/metabolismo , Osteocitos/citología , Transducción de Señal/fisiología , Animales , Comunicación Celular/fisiología , Conexina 43/genética , Humanos , Osteoblastos/citologíaRESUMEN
BACKGROUND: Recent work has shown that the gap junction protein connexin43 (Cx43) is upregulated in cells of the joint during osteoarthritis (OA). Here we examined if the OA-associated increase in Cx43 expression impacts the function of synovial fibroblasts by contributing to the production of catabolic and inflammatory factors that exacerbate joint destruction in arthritic disease. METHODS: Using rabbit and human synovial fibroblast cell lines, we examined the effects of Cx43 overexpression and Cx43 siRNA-mediated knockdown on the gene expression of OA-associated matrix metalloproteinases (MMP1 and MMP13), aggrecanases (ADAMTS4 and ADAMTS5), and inflammatory factors (IL1, IL6 and PTGS2) by quantitative real time RT-PCR. We examined collagenase activity in conditioned media of cultured synovial cells following Cx43 overexpression. Lastly, we assessed the interplay between Cx43 and the NFκB cascade by western blotting and gene expression studies. RESULTS: Increasing Cx43 expression enhanced the gene expression of MMP1, MMP13, ADAMTS4, ADAMTS5, IL1, IL6 and PTGS2 and increased the secretion of collagenases into conditioned media of cultured synovial fibroblasts. Conversely, knockdown of Cx43 decreased expression of many of these catabolic and inflammatory genes. Modulation of Cx43 expression altered the phosphorylation of the NFκB subunit, p65, and inhibition of NFκB with chemical inhibitors blocked the effects of increased Cx43 expression on the mRNA levels of a subset of these catabolic and inflammatory genes. CONCLUSIONS: Increasing or decreasing Cx43 expression alone was sufficient to alter the levels of catabolic and inflammatory genes expressed by synovial cells. The NFκB cascade mediated the effect of Cx43 on the expression of a subset of these OA-associated genes. As such, Cx43 may be involved in joint pathology during OA, and targeting Cx43 expression or function may be a viable therapeutic strategy to attenuate the catabolic and inflammatory environment of the joint during OA.
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Conexina 43/biosíntesis , Conexina 43/genética , Fibroblastos/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , Membrana Sinovial/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Conejos , RatasRESUMEN
3D bioprinting holds great promise for meeting the increasing need for transplantable tissues and organs. However, slow printing, interlayer mixing, and the extended exposure of cells to non-physiological conditions in thick structures still hinder clinical applications. Here the DeepFreeze-3D (DF-3D) procedure and bioink for creating multilayered human-scale tissue mimetics is presented for the first time. The bioink is tailored to support stem cell viability, throughout the rapid freeform DF-3D biofabrication process. While the printer nozzle is warmed to room temperature, each layer solidifies at contact with the stage (-80 °C), or the subsequent layers, ensuring precise separation. After thawing, the encapsulated stem cells remain viable without interlayer mixing or delamination. The composed cell-laden constructs can be cryogenically stored and thawed when needed. Moreover, it is shown that under inductive conditions the stem cells differentiate into bone-like cells and grow for months after thawing, to form large tissue-mimetics in the scale of centimeters. This is important, as this approach allows the generation and storage of tissue mimetics in the size and thickness of human tissues. Therefore, DF-3D biofabrication opens new avenues for generating off-the-shelf human tissue analogs. It further holds the potential for regenerative treatments and for studying tissue pathologies caused by disease, tumor, or trauma.
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Bioimpresión , Ingeniería de Tejidos , Humanos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Impresión Tridimensional , Bioimpresión/métodos , Bioingeniería , Células MadreRESUMEN
The most common and severe form of muscular dystrophy is Duchenne muscular dystrophy (DMD), a disorder caused by the absence of dystrophin, a structural protein found on the cytoplasmic surface of the sarcolemma of striated muscle fibres. Considerable attention has been dedicated to studying myofibre damage and muscle plasticity, but there is little information to determine if damage from contraction-induced injury occurs at or near the nerve terminal axon. We used α-bungarotoxin to compare neuromuscular junction (NMJ) morphology in healthy (wild-type, WT) and dystrophic (mdx) mouse quadriceps muscles and evaluated transcript levels of the post-synaptic muscle-specific kinase signalling complex. Our focus was to study changes in NMJs after injury induced with an established in vivo animal injury model. Neuromuscular transmission, electromyography (EMG), and NMJ morphology were assessed 24 h after injury. In non-injured muscle, muscle-specific kinase expression was significantly decreased in mdx compared to WT. Injury resulted in a significant loss of maximal torque in WT (39 ± 6%) and mdx (76 ± 8%) quadriceps, but significant changes in NMJ morphology, neuromuscular transmission and EMG data were found only in mdx following injury. Compared with WT mice, motor end-plates of mdx mice demonstrated less continuous morphology, more disperse acetylcholine receptor aggregates and increased number of individual acetylcholine receptor clusters, an effect that was exacerbated following injury. Neuromuscular transmission failure increased and the EMG measures decreased after injury in mdx mice only. The data show that eccentric contraction-induced injury causes morphological and functional changes to the NMJs in mdx skeletal muscle, which may play a role in excitation-contraction coupling failure and progression of the dystrophic process.
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Contracción Isométrica , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Unión Neuromuscular/fisiopatología , Animales , Axones/ultraestructura , Bungarotoxinas , Distrofina/genética , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Placa Motora/citología , Distrofia Muscular de Duchenne/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , ARN Mensajero/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/metabolismo , TorqueRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by bone erosion and cartilage destruction in the joints. Many of the conventional antiarthritic drugs are effective in suppressing inflammation, but they do not offer protection against bone damage. Furthermore, the prolonged use of these drugs is associated with severe adverse reactions. Thus, new therapeutic agents that can control both inflammation and bone damage but with minimal side effects are sought. Celastrus is a Chinese herb that has been used for centuries in folk medicine for the treatment of various inflammatory diseases. However, its utility for protection against inflammation-induced bone damage in arthritis and the mechanisms involved therein have not been examined. We tested celastrus and its bioactive component celastrol for this attribute in the adjuvant-induced arthritis model of RA. The treatment of arthritic rats with celastrus/celastrol suppressed inflammatory arthritis and reduced bone and cartilage damage in the joints as demonstrated by histology and bone histomorphometry. The protective effects against bone damage are mediated primarily via the inhibition of defined mediators of osteoclastic bone remodeling (e.g. receptor activator of nuclear factor-κB ligand (RANKL)), the deviation of RANKL/osteoprotegerin ratio in favor of antiosteoclastic activity, and the reduction in osteoclast numbers. Furthermore, both the upstream inducers (proinflammatory cytokines) and the downstream effectors (MMP-9) of the osteoclastogenic mediators were altered. Thus, celastrus and celastrol controlled inflammation-induced bone damage by modulating the osteoimmune cross-talk. These natural products deserve further consideration and evaluation as adjuncts to conventional therapy for RA.