RESUMEN
Mechanical signals regulate a multitude of cell functions and ultimately govern fibrous tissue growth, maintenance and repair. Such mechanotransduction processes often involve modulation of intracellular calcium concentration ([Ca2+]i). However, most studies interrogate these responses in cells in simplified culture systems, thereby removing potentially important inputs from the native extracellular microenvironment. The objective of this study was to test the hypothesis that the intracellular calcium response of meniscus fibrochondrocytes (MFCs) is dependent on both the microenvironmental context in which this perturbation is applied and on the tensile deformation. Using a custom micro-mechanical tester mounted on a confocal microscope, intracellular calcium activity in MFCs in response to incremental tissue strains (0, 3, 6 and 9 %) was monitored in situ (i.e., in the native tissues) on MFC-seeded aligned scaffolds and MFC-seeded silicone membranes. The [Ca2+]i regulation by MFCs within the native meniscus tissue microenvironment was considerably different from [Ca2+]i regulation by MFCs on either aligned nanofibrous scaffolds or flat silicone membranes. Additionally, increasing levels of tensile deformation resulted in a greater number of responding cells, both in situ and in vitro, while having no effects on temporal characteristics of [Ca2+]i signalling. Collectively, these findings have significant implications for mechanobiology of load-bearing fibrous tissues and their responses to injury and degeneration. In addition, from a tissue engineering perspective, the findings establish cellular benchmarks for maturing engineered constructs, where native tissue-like calcium mechano-regulation may be an important outcome parameter to achieve mechanical functionality comparable to native tissue.
Asunto(s)
Señalización del Calcio , Microambiente Celular , Condrocitos/citología , Condrogénesis , Meniscos Tibiales/citología , Resistencia a la Tracción , Animales , Bovinos , Condrocitos/metabolismo , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
This study aimed to understand the role of Cav1.3, one of the four L-type voltage sensitive calcium channels (VSCC) alpha(1) subunits, in the skeletal response to mechanical loading and intermittent PTH treatment. The Cav1.3 mRNA is expressed in osteoblasts. The Cav1.3 mRNA level in male wild type mice is higher than those in female. Loss of Cav1.3 resulted in a smaller skeleton in male mice as indicated by significantly lower body weight, less bone mineral content and smaller cross-sectional area of femoral midshaft. However, the osteogenic response to mechanical loading of the ulna was normal in Cav1.3(-/-) compared to the normal control mice. Male mice Cav1.3(-/-) were then treated daily with PTH at a dose of 40 microg/kg. A 6-week course of intermittent PTH treatment enhanced bone mineral content and mechanical strength equally in wild type control and Cav1.3 null mice. We also found that Cav1.2 subunit significantly increases in the absence of Cav1.3 gene. In conclusion, Cav1.3 is involved in bone metabolism, especially in male mice. Cav1.3 does not mediate osteoblast response to mechanical loading and PTH. Our data suggest that Cav1.1 and Cav1.2 subunits may substitute for Cav1.3 to maintain bone response to mechanical loading.
Asunto(s)
Densidad Ósea/fisiología , Canales de Calcio Tipo L/genética , Fémur/fisiología , Hormona Paratiroidea/farmacología , Cúbito/fisiología , Análisis de Varianza , Animales , Fenómenos Biomecánicos , Densidad Ósea/efectos de los fármacos , Fémur/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Estrés Mecánico , Cúbito/efectos de los fármacosRESUMEN
Bone cells are organized into an interconnected network, which extends from the osteocytes within bone to the osteoblasts and lining cells on the bone surfaces. There is experimental evidence suggesting that bone tissue exhibits basic properties of short- and long-term memory. An analogy might be made between the bone cell network and neuronal systems. For instance, recent studies suggest that the neurotransmitter glutamate may play a role in cell-to-cell communication among bone cells. Glutamate is a key neurotransmitter involved in learning and memory in reflex loops and the hippocampus. The simplest forms of memory include habituation (desensitization) and sensitization. It is argued that bone cells exhibit habituation to repeated mechanical stimuli and sensitization to mechanical loading by parathyroid hormone (PTH). Acquired long-term memory of a mechanical loading environment may influence the responsiveness of bone tissue to external stimuli. For instance, bone tissue from the skull shows markedly different responses to several stimuli, e.g., mechanical loading, disuse, and PTH, compared with long bones. We speculate that the history of weight bearing imparts long-term cellular memory to the bone cell network that modulates the cellular response to a wide variety of stimuli.
Asunto(s)
Huesos/citología , Red Nerviosa/citología , Osteocitos/citología , Animales , Huesos/efectos de los fármacos , Huesos/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Ácido Glutámico/fisiología , Humanos , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Osteocitos/efectos de los fármacos , Osteocitos/fisiología , Hormona Paratiroidea/farmacología , Estrés Mecánico , Soporte de Peso/fisiologíaRESUMEN
Osteoblasts respond to both fluid shear and parathyroid hormone (PTH) with a rapid increase in intracellular calcium concentration ([Ca2+]i). Because both stimuli modulate the kinetics of the mechanosensitive cation channel (MSCC), we postulated PTH would enhance the [Ca2+]i response to fluid shear by increasing the sensitivity of MSCCs. After a 3-minute preflow at 1 dyne/cm2, MC3T3-E1 cells were subjected to various levels of shear and changes in [Ca2+]i were assessed using Fura-2. Pretreatment with 50 nM bovine PTH(1-34) [bPTH(1-34)] significantly enhanced the shear magnitude-dependent increase in [Ca2+]i. Gadolinium (Gd3+), an MSCC blocker, significantly inhibited the mean peak [Ca2+]i response to shear and shear + bPTH(1-34). Nifedipine (Nif), an L-type voltage-sensitive Ca2+ channel (VSCC) blocker, also significantly reduced the [Ca2+]i response to shear + bPTH(1-34), but not to shear alone, suggesting VSCC activation plays an interactive role in the action of these stimuli together. Activation of either the protein kinase C (PKC) or protein kinase A (PKA) pathways with specific agonists indicated that PKC activation did not alter the Ca2+ response to shear, whereas PKA activation significantly increased the [Ca2+]i response to lower magnitudes of shear. bPTH(1-34), which activates both pathways, induced the greatest [Ca2+]i response at each level of shear, suggesting an interaction of these pathways in this response. These data indicate that PTH significantly enhances the [Ca2+]i response to shear primarily via PKA modulation of the MSCC and VSCC.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Células 3T3 , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación del Canal Iónico , Ratones , Osteoblastos/metabolismo , Proteína Quinasa C/metabolismoRESUMEN
Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca(2+)](i). Although PTH did not significantly change [Ca(2+)](i) levels, flow and PTH did produce a significantly greater [Ca(2+)](i) response and increased the number of responding cells than is found in fluid shear alone. The [Ca(2+)](i) response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca(2+)](i) by modulating the mechanosensitive channel.
Asunto(s)
Matriz Extracelular/fisiología , Osteoblastos/fisiología , Hormona Paratiroidea , Animales , Calcio/metabolismo , Línea Celular , Ciclooxigenasa 2 , Matriz Extracelular/efectos de los fármacos , Gadolinio/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Hormona Paratiroidea/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/metabolismo , Estrés Mecánico , Soporte de PesoRESUMEN
Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.
Asunto(s)
Calcio/metabolismo , Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Osteoblastos/metabolismo , Células 3T3 , Actinas/biosíntesis , Actinas/genética , Animales , Señalización del Calcio/efectos de los fármacos , Ciclooxigenasa 2 , Citoesqueleto/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Genes fos , Isoenzimas/biosíntesis , Isoenzimas/genética , Ratones , Osteoblastos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Mecánico , Tapsigargina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Essential or primary hypertension is a multifactorial disease that is expressed as a result of complex interactions between genes and environmental influences. Several mutations in many different proteins are associated with expression of hypertension, including abnormalities in the epithelial sodium channel (ENaC) found in absorptive organs (i.e., distal colon, distal tubule of the nephron). Some of these mutations result in structural and/or functional alterations in ENaC-mediated Na+ entry in epithelia responsible for fluid and electrolyte balance and are associated with expression of hypertension. Studies support the notion that there is a link between ENaC and hypertension of both the monogenic (single gene mutation) and primary or essential type (a multifactorial disease). Alterations of other aspects of the environment of absorptive cells (e.g., hyperinsulinemia, hyperaldosteronemia, high plasma cortisol, high plasma Na+) have also been shown to elicit hyperabsorption of Na+ via ENaC and therefore could contribute significantly to expression of hypertension in people with intermediate phenotypes. This article describes an initial study in which the effects of an environmental factor, extracellular levels of insulin, on ENaC were examined in a normal kidney cell model. Electrophysiologic techniques revealed that ENaC density rapidly increased in response to addition of insulin to the basolateral bath. This autoregulatory recruitment of Na+ total channel density masked a slight decrease in open channel probability. Insulin's effect on ENaC function and implications on fluid and electrolyte balance and expression of primary hypertension is discussed.
Asunto(s)
Modelos Animales de Enfermedad , Epitelio/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hipertensión/inducido químicamente , Hipertensión/genética , Hipoglucemiantes/efectos adversos , Insulina/efectos adversos , Túbulos Renales Distales/citología , Mutación/genética , Canales de Sodio/efectos de los fármacos , Animales , Recuento de Células , Células Cultivadas , Homeostasis/efectos de los fármacos , Ranidae , Factores de Riesgo , Canales de Sodio/ultraestructura , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.
Asunto(s)
Citoesqueleto/fisiología , Integrinas/fisiología , Osteoblastos/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/enzimología , Actinina/farmacología , Actinas/fisiología , Animales , Adhesión Celular/fisiología , Línea Celular , Ciclooxigenasa 2 , Citocalasina D/farmacología , Proteínas de Unión al GTP/genética , Expresión Génica/efectos de los fármacos , Integrina beta1/metabolismo , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Ratones , Osteoblastos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Estrés Mecánico , Proteína de Unión al GTP rhoBRESUMEN
Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.
Asunto(s)
Canales de Calcio/fisiología , Calcio/fisiología , Osteoblastos/fisiología , Huesos/química , Huesos/citología , Huesos/fisiología , Membrana Celular/química , Membrana Celular/fisiología , Humanos , Osteoblastos/química , Osteoblastos/citología , Transducción de SeñalRESUMEN
Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73-112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 microA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6-20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging approximately 0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.
Asunto(s)
Aldosterona/farmacología , Amilorida/farmacología , Corticosterona/farmacología , Canales de Sodio/fisiología , Amilorida/análogos & derivados , Técnicas de Cultivo de Célula/métodos , División Celular , Línea Celular , Medios de Cultivo , Conductividad Eléctrica , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Probabilidad , Bloqueadores de los Canales de Sodio , Canales de Sodio/biosíntesisRESUMEN
Mechanical force applied to bone produces two localized mechanical signals on the cell: deformation of the extracellular matrix (substrate strain) and extracellular fluid flow. To study the effects of these stimuli on osteoblasts, MC3T3-E1 cells were grown on type I collagen-coated plastic plates and subjected to four-point bending. This technique produces uniform levels of physiological strain and fluid forces on the cells. Each of these parameters can be varied independently. Osteopontin (OPN) mRNA expression was used to assess the anabolic response of MC3T3-E1 cells. When fluid forces were low, neither strain magnitude nor strain rate was correlated with OPN expression. However, higher-magnitude fluid forces significantly increased OPN message levels independently of the strain magnitude or rate. These data indicate that fluid forces, and not mechanical stretch, influence OPN expression in osteoblasts and suggest that fluid forces induced by extracellular fluid flow within the bone matrix may play an important role in bone formation in response to mechanical loading.
Asunto(s)
Matriz Extracelular/fisiología , Osteoblastos/fisiología , Transducción de Señal/fisiología , Células 3T3 , Animales , Colágeno , Espacio Extracelular/fisiología , Cinética , Ratones , Osteopontina , Fosfoproteínas/biosíntesis , ARN Mensajero/biosíntesis , Sialoglicoproteínas/biosíntesis , Estrés Mecánico , Transcripción GenéticaRESUMEN
We previously demonstrated electrophysiologically that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] shifts the activation threshold of L-type Ca2+ channels in osteoblasts toward the resting potential and prolongs mean open time. Presently, we used single-cell Ca2+ imaging to study the combined effects of 1,25(OH)2D3 and parathyroid hormone (PTH) during generation of Ca2+ transients in fura 2-loaded MC3T3-E1 cells. Pretreatment with 1,25(OH)2D3 concentrations, which alone did not produce Ca2+ transients, consistently enhanced Ca2+ responses to PTH. Enhancement was dose dependent over the range of 1 to 10 nM and was blocked by pretreatment with 5 microM nitrendipine during pretreatment. A 1,25(OH)2D3 analog that activates L-type channels and shifts their activation threshold also enhanced PTH responses. In contrast, an analog devoid of membrane Ca2+ effects did not enhance PTH-induced Ca2+ transients. The PTH-induced Ca2+ transient involved activation of a dihydropyridine-insensitive cation channel that was inhibited by Gd3+. Together, these data suggest that 1,25(OH)2D3 increases osteoblast responsiveness to PTH through rapid modification of L-type Ca2+ channel gating properties, whose activation enhances Ca2+ entry through other channels such as the PTH-responsive, Gd(3+)-sensitive cation channel.
Asunto(s)
Calcitriol/farmacología , Canales de Calcio/fisiología , Calcio/metabolismo , Osteoblastos/fisiología , Hormona Paratiroidea/farmacología , Células 3T3 , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L , Diferenciación Celular , Sinergismo Farmacológico , Gadolinio/farmacología , Cinética , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Factores de TiempoRESUMEN
By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.
Asunto(s)
Canales de Calcio/fisiología , Osteoblastos/fisiología , Equilibrio Hidroelectrolítico , Animales , Secuencia de Bases , Tamaño de la Célula , Conductividad Eléctrica , Activación del Canal Iónico , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/química , Osteosarcoma , Técnicas de Placa-Clamp , Periodicidad , Ratas , Estrés Mecánico , Células Tumorales CultivadasRESUMEN
Mechanotransduction plays a crucial role in the physiology of many tissues including bone. Mechanical loading can inhibit bone resorption and increase bone formation in vivo. In bone, the process of mechanotransduction can be divided into four distinct steps: (1) mechanocoupling, (2) biochemical coupling, (3) transmission of signal, and (4) effector cell response. In mechanocoupling, mechanical loads in vivo cause deformations in bone that stretch bone cells within and lining the bone matrix and create fluid movement within the canaliculae of bone. Dynamic loading, which is associated with extracellular fluid flow and the creation of streaming potentials within bone, is most effective for stimulating new bone formation in vivo. Bone cells in vitro are stimulated to produce second messengers when exposed to fluid flow or mechanical stretch. In biochemical coupling, the possible mechanisms for the coupling of cell-level mechanical signals into intracellular biochemical signals include force transduction through the integrin-cytoskeleton-nuclear matrix structure, stretch-activated cation channels within the cell membrane, G protein-dependent pathways, and linkage between the cytoskeleton and the phospholipase C or phospholipase A pathways. The tight interaction of each of these pathways would suggest that the entire cell is a mechanosensor and there are many different pathways available for the transduction of a mechanical signal. In the transmission of signal, osteoblasts, osteocytes, and bone lining cells may act as sensors of mechanical signals and may communicate the signal through cell processes connected by gap junctions. These cells also produce paracrine factors that may signal osteoprogenitors to differentiate into osteoblasts and attach to the bone surface. Insulin-like growth factors and prostaglandins are possible candidates for intermediaries in signal transduction. In the effector cell response, the effects of mechanical loading are dependent upon the magnitude, duration, and rate of the applied load. Longer duration, lower amplitude loading has the same effect on bone formation as loads with short duration and high amplitude. Loading must be cyclic to stimulate new bone formation. Aging greatly reduces the osteogenic effects of mechanical loading in vivo. Also, some hormones may interact with local mechanical signals to change the sensitivity of the sensor or effector cells to mechanical load.
Asunto(s)
Densidad Ósea/fisiología , Remodelación Ósea/fisiología , Huesos/fisiología , Transducción de Señal/fisiología , Estrés Mecánico , HumanosRESUMEN
One physiologic consequence of extended periods of weightlessness is the rapid loss of bone mass associated with skeletal unloading. Conversely, mechanical loading has been shown to increase bone formation and stimulate osteoblastic function. The mechanisms underlying mechanotransduction, or how the osteoblast senses and converts biophysical stimuli into cellular responses has yet to be determined. For non-innervated mechanosensitive cells like the osteoblast, mechanotransduction can be divided into four distinct phases: 1) mechanocoupling, or the characteristics of the mechanical force applied to the osteoblast, 2) biochemical coupling, or the mechanism through which mechanical strain is transduced into a cellular biochemical signal, 3) transmission of signal from sensor to effector cell and 4) the effector cell response. This review examines the characteristics of the mechanical strain encountered by osteoblasts, possible biochemical coupling mechanisms, and how the osteoblast responds to mechanical strain. Differences in osteoblastic responses to mechanical strain are discussed in relation to the types of strain encountered and the possible transduction pathways involved.
Asunto(s)
Desmineralización Ósea Patológica/fisiopatología , Huesos/fisiología , Osteoblastos/fisiología , Transducción de Señal/fisiología , Animales , Fenómenos Biomecánicos , Huesos/citología , Huesos/fisiopatología , Integrinas/fisiología , Activación del Canal Iónico/fisiología , Osteoblastos/citología , Ratas , Vuelo Espacial , Estrés Mecánico , IngravidezRESUMEN
Exposure of osteosarcoma cell lines to chronic intermittent strain increases the activity of mechano-sensitive cation (SA-cat) channels. The impact of mechano-transduction on osteoblast function has not been well studied. We analyzed the expression and production of bone matrix proteins in human osteoblast-like osteosarcoma cells, OHS-4, in response to chronic intermittent mechanical strain. The OHS-4 cells exhibit type I collagen production, 1,25-Dihydroxyvitamin D-inducible osteocalcin, and mineralization of the extracellular matrix. The matrix protein message level was determined from total RNA isolated from cells exposed to 1-4 days of chronic intermittent strain. Northern analysis for type I collagen indicated that strain increased collagen message after 48 h. Immunofluorescent labeling of type I collagen demonstrated that secretion was also enhanced with mechanical strain. Osteopontin message levels were increased several-fold by the application of mechanical load in the absence of vitamin D, and the two stimuli together produced an additive effect. Osteocalcin secretion was also increased with cyclic strain. Osteocalcin levels were not detectable in vitamin D-untreated control cells. However, after 4 days of induced load, significant levels of osteocalcin were observed in the medium. With vitamin D present, osteocalcin levels were 4 times higher in the medium of strained cells compared to nonstrained controls. We conclude that mechanical strain of osteoblast-like cells is sufficient to increase the transcription and secretion of matrix proteins via mechano-transduction without hormonal induction.
Asunto(s)
Colágeno/biosíntesis , Osteocalcina/biosíntesis , Osteosarcoma/metabolismo , Sialoglicoproteínas/biosíntesis , Northern Blotting , Calcitriol/farmacología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Osteocalcina/antagonistas & inhibidores , Osteopontina , Osteosarcoma/patología , ARN/análisis , Transducción de Señal , Estrés Mecánico , Células Tumorales CultivadasRESUMEN
The effects of chronic, intermittent strain on the mechanosensitive cation (SA-cat) channels in UMR-106.01 osteoblast-like osteosarcoma cells were studied using patch-clamp techniques. Chronically strained cells demonstrated significantly larger increases in whole cell conductance when subjected to additional mechanical strain than nonstrained controls (69.0 +/- 15.1 vs. 14.1 +/- 3.1%; P < 0.001). This increase could be blocked by the SA-cat channel inhibitor, gadolinium, and corresponded to a three- to fivefold increase in SA-cat channel activity. Chronic strain increased the number of open channels in response to stretch and induced spontaneous SA-cat channel activity in 33% of the patches of strained cells. Graded increases in negative patch pressure demonstrated that SA-cat channels in chronically strained cells were activated at significantly lower levels of mechanical perturbation than nonstrained controls. These data suggest that chronic, cyclic strain reduces the activation threshold of the SA-cat channel and further strengthen our hypothesis that this channel may act as a mechanotransducer for the activation of bone remodeling by physical strain.
Asunto(s)
Canales Iónicos/fisiología , Mecanorreceptores/fisiología , Osteoblastos/fisiología , Cationes , Conductividad Eléctrica , Gadolinio/farmacología , Osteosarcoma , Técnicas de Placa-Clamp , Estrés Mecánico , Células Tumorales CultivadasRESUMEN
Cell-attached patches of membrane of osteoblast-like cells UMR-106.01 respond to bath application of parathyroid hormone (PTH) with an increase in the average activity, as well as the single channel conductance, of a stretch-activated non-selective cation channel. Correlations with whole cell membrane potential and conductance changes are considered.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Hormona Paratiroidea/fisiología , Animales , Conductividad Eléctrica , Mecanorreceptores/fisiología , Osteosarcoma , Ratas , Células Tumorales CultivadasRESUMEN
By using a selective medium, pharyngeal colonization with gram-negative rod (GNR) bacteria was determined in a cohort of 49 normal infants monitored from birth to 6 months of age. Culture swabs were diluted in 1 ml of saline for quantitation. The prevalence of GNR in the first 72 h of life was 8% and rose to 29% during the first month, 52% at 2.5 months, 67% at 4.5 months, and 62% at 6 to 7 months. Colonization was with substantial numbers of organisms, generally greater than 100 colonies per ml and frequently greater than 1,000 colonies per ml. The most common species were Klebsiella species, Escherichia coli, Enterobacter species, and Acinetobacter anitratus. Fewer infants who were breast fed rather than formula fed at the time of culture harbored GNR (26 versus 45%, P less than 0.05). The point prevalence of pharyngeal GNR colonization in our special care nursery was 12 of 47 (26%), which was found to be similar to that of age-matched normal infants. GNR carriage in normal infants does not appear to be a residual of organisms acquired at birth, and interpretations of GNR carriage in ill or hospitalized infants should be evaluated by comparison with these data in healthy infants.
Asunto(s)
Bacterias Aerobias Gramnegativas/crecimiento & desarrollo , Faringe/microbiología , Factores de Edad , Alimentación con Biberón , Lactancia Materna , Recuento de Colonia Microbiana , Humanos , Lactante , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , Estudios Longitudinales , Estudios Prospectivos , Encuestas y CuestionariosRESUMEN
This study tests the hypothesis, in A6 epithelia, that 1) corticosterone stimulates active Na+ transport (short-circuit current, Isc) by an additional receptor mechanism to the type I (mineralocorticoid) and type II (glucocorticoid) mechanisms shared with aldosterone (Aldo) and 2) that the agonist may be 6 beta-OH-corticosterone made in the effector cell. The dose-response relationship of corticosterone at 24 h resolves into two components, by curve fitting, with a 50% effective concentration (EC50) for 10% of maximum Isc stimulation of 2 X 10(-9) M and an EC50 for the other 90% of 3 X 10(-7) M. The EC50 of the smaller component correlates with the apparent dissociation constant (K'd) of corticosterone for high affinity (type II) nuclear binding sites shared with Aldo. In unlabeled analogue competition studies Aldo and corticosterone displaced nuclear binding equally below 10(-8) M [3H]corticosterone, indicating only shared sites. However, nonshared saturable sites (displaced by corticosterone but not by Aldo) were found at [3H]-corticosterone concentrations above 10(-8) M. Concentration-binding curves performed with [3H]corticosterone, in presence of 1,000 X Aldo to displace shared sites, revealed a single class of binding sites with a half-maximal saturation of 2 X 10(-7) M, which is quite similar to the EC50 of the lower affinity component of Isc stimulation by corticosterone at 24 h. Reversed phase high-pressure liquid chromatography of nuclear extracts indicates that the saturable component of bound [3H] was 6 beta-OH-[3H]corticosterone derived from [3H]corticosterone. Thus, A6 cells metabolize corticosterone to 6 beta-OH-corticosterone, which in turn occupies lower-affinity receptors not shared with Aldo or corticosterone, to mediate most of the active Na+ transport stimulation by corticosterone.