Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage.

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.

Sodium-calcium exchanger 1 regulates epithelial cell migration via calcium-dependent extracellular signal-regulated kinase signaling.

Na(+)/Ca(2+) exchanger-1 (NCX1) is a major calcium extrusion mechanism in renal epithelial cells enabling the efflux of one Ca(2+) ion and the influx of three Na(+) ions. The gradient for this exchange activity is provided by Na,K-ATPase, a hetero-oligomer consisting of a catalytic α-subunit and a regulatory ß-subunit (Na,K-ß) that also functions as a motility and tumor suppressor. We showed earlier that mice with heart-specific ablation (KO) of Na,K-ß had a specific reduction in NCX1 protein and were ouabain-insensitive. Here, we demonstrate that Na,K-ß associates with NCX1 and regulates its localization to the cell surface. Madin-Darby canine kidney cells with Na,K-ß knockdown have reduced NCX1 protein and function accompanied by 2.1-fold increase in free intracellular calcium and a corresponding increase in the rate of cell migration. Increased intracellular calcium up-regulated ERK1/2 via calmodulin-dependent activation of PI3K. Both myosin light chain kinase and Rho-associated kinase acted as mediators of ERK1/2-dependent migration. Restoring NCX1 expression in ß-KD cells reduced migration rate and ERK1/2 activation, suggesting that NCX1 functions downstream of Na,K-ß in regulating cell migration. In parallel, inhibition of NCX1 by KB-R7943 in Madin-Darby canine kidney cells, LLC-PK1, and human primary renal epithelial cells (HREpiC) increased ERK1/2 activation and cell migration. This increased migration was associated with high myosin light chain phosphorylation by PI3K/ERK-dependent mechanism in HREpiC cells. These data confirm the role of NCX1 activity in regulating renal epithelial cell migration.

Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.

The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

A mutation in TRPV4 results in altered chondrocyte calcium signaling in severe metatropic dysplasia.

Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a polymodal modulated non-selective cation channel required for normal development and maintenance of bone and cartilage. Heterozygous mutations of this channel cause a variety of channelopathies, including metatropic dysplasia (MD). We analyzed the effect of a novel TRPV4 mutation c.2398G>A, p.Gly800Asp on intracellular calcium ([Ca(2+) ]i ) regulation in chondrocytes and compared this response to chondrocytes with a frequently observed mutation, c.2396C>T, p.Pro799Leu. We observed temperature-dependent [Ca(2+) ]i oscillations in both intact and MD chondrocytes however, MD mutations exhibited increased peak magnitudes of [Ca(2+) ]i during oscillations. We also found increased baseline [Ca(2+) ]i in MD primary cells, as well as increased [Ca(2+) ]i response to either hypotonic swelling or the TRVP4-specific agonist, GSK1016790A. Oscillations and stimulation responses were blocked with the TRPV4-specific antagonist, GSK205. Analysis of [Ca(2+) ]i response kinetics showed that MD chondrocytes had increased frequency of temperature-sensitive oscillations, and the magnitude and duration of [Ca(2+) ]i responses to given stimuli. Duration of the response of the p.Gly800Asp mutation to stimulation was greater than for the p.Pro799Leu mutation. These experiments show that this region of the channel is essential for proper [Ca(2+) ]i regulation. These studies of primary cells from patients show how both mutant and WT TRPV4 channels regulate cartilage and bone development. © 2015 Wiley Periodicals, Inc.

P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow.

Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca(2+) concentration ([Ca(2+)]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.

Exaggerated exercise pressor reflex in adults with moderately elevated systolic blood pressure: role of purinergic receptors.

The neurocirculatory responses to exercise are exaggerated in hypertension, increasing cardiovascular risk, yet the mechanisms remain incompletely understood. The aim of this study was to examine the in vitro effectiveness of pyridoxal-5-phosphate as a purinergic (P2) receptor antagonist in isolated murine dorsal root ganglia (DRG) neurons and the in vivo contribution of P2 receptors to the neurocirculatory responses to exercise in older adults with moderately elevated systolic blood pressure (BP). In vitro, pyridoxal-5-phosphate attenuated the ATP-induced increases in [Ca(2+)](i) (73 ± 15 vs. 11 ± 3 nM; P < 0.05). In vivo, muscle sympathetic nerve activity (MSNA; peroneal microneurography) and arterial BP (Finometer) were assessed during exercise pressor reflex activation (static handgrip followed by postexercise ischemia; PEI) during a control trial (normal saline) and localized P2 receptor blockade (pyridoxal-5-phosphate). Compared with normotensive adults (63 ± 2 yr, 117 ± 2/70 ± 2 mmHg), adults with moderately elevated systolic BP (65 ± 1 yr, 138 ± 5/79 ± 3 mmHg) demonstrated greater increases in MSNA and BP during handgrip and PEI. Compared with the control trial, local antagonism of P2 receptors during PEI partially attenuated MSNA (39 ± 4 vs. 34 ± 5 bursts/min; P < 0.05) in adults with moderately elevated systolic BP. In conclusion, these data demonstrate pyridoxal-5-phosphate is an effective P2 receptor antagonist in isolated DRG neurons, which are of particular relevance to the exercise pressor reflex. Furthermore, these findings indicate that exercise pressor reflex function is exaggerated in older adults with moderately elevated systolic BP and further suggest a modest role of purinergic receptors in evoking the abnormally large reflex-mediated increases in sympathetic activity during exercise in this clinical population.

Prostaglandin E2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 cells in a PKA-dependent manner.

The effect of prostaglandin E2 (PGE2) on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regulates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteoblasts in the presence of fluid shear stress (FSS), which could better uncover the anabolic effect of PGE2 in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A (PKA) pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 min significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells' calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the 'reset' process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone formation as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases.

CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.

Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.

Overexpression of the VSSC-associated CAM, ß-2, enhances LNCaP cell metastasis associated behavior.

BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer death in American men. This is due largely to the "silent" nature of the disease until it has progressed to a highly metastatic and castrate resistant state. Voltage sensitive sodium channels (VSSCs) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two ß subunits. The ß-subunits modulate surface expression and gating kinetics of the channels but also have inherent cell adhesion molecule (CAM) functions. We hypothesize that PCa cells use VSSC ß-subunits as CAMs during PCa progression and metastasis. METHODS: We overexpressed the beta-2 isoform as a C-terminal fusion protein with enhanced cyan fluorescence protein (ECFP) in the weakly metastatic LNCaP cells. The effect of beta-2 overexpression on cell morphology was examined using confocal microscopy while metastasis-associated behavior was tested by performing several in vitro metastatic functional assays and in vivo subcutaneous tumor studies. RESULTS: We found that cells overexpressing beta-2 (2BECFP) converted to a bipolar fibroblastic morphology. 2BECFP cells were more adhesive than control (ECFP) to vitronectin (twofold) and Matrigel® (1.3-fold), more invasive through Matrigel® (3.6-fold in 72 hr), and had enhanced migration (2.1-fold in 96 hr) independent of proliferation in wound-healing assays. In contrast, 2BECFP cells have a reduced tumor-take and tumor volume in vivo even though the overexpression of beta-2 was maintained. CONCLUSIONS: Functional overexpression of VSSC ß-subunits in PCa may be one mechanism leading to increased metastatic behavior while decreasing the ability to form localized tumor masses.

Purinergic signaling is required for fluid shear stress-induced NF-κB translocation in osteoblasts.

Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-κB. We examined whether this process was under the control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-κB inhibitory protein IκBα and exhibited cytosolic localization of NF-κB. Under fluid shear stress, IκBα levels decreased, and concomitant nuclear localization of NF-κB was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in IκBα, and NF-κB remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X(7) receptor antagonists, indicating that the P2X(7) receptor is responsible for fluid shear-stress-induced IκBα degradation and nuclear accumulation of NF-κB. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced IκBα degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X(7)-generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-κB activity through the P2Y(6) and P2X(7) receptor.

Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4.

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

Hierarchically structured, hyaluronic acid-based hydrogel matrices via the covalent integration of microgels into macroscopic networks.

We aimed to develop biomimetic hydrogel matrices that not only exhibit structural hierarchy and mechanical integrity, but also present biological cues in a controlled fashion. To this end, photocrosslinkable, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized via an inverse emulsion crosslinking process followed by chemical modification with glycidyl methacrylate (GMA). HA modified with GMA (HA-GMA) was employed as the soluble macromer. Macroscopic hydrogels containing covalently integrated hydrogel particles (HA-c-HGP) were prepared by radical polymerization of HA-GMA in the presence of crosslinkable HGPs. The covalent linkages between the hydrogel particles and the secondary HA matrix resulted in the formation of a diffuse, fibrilar interface around the particles. Compared to the traditional bulk gels synthesized by photocrosslinking of HA-GMA, these hydrogels exhibited a reduced sol fraction and a lower equilibrium swelling ratio. When tested under uniaxial compression, the HA-c-HGP gels were more pliable than the HA-p-HGP gels and fractured at higher strain than the HA-GMA gels. Primary bovine chondrocytes were photoencapsulated in the HA matrices with minimal cell damage. The 3D microenvironment created by HA-GMA and HA HGPs not only maintained the chondrocyte phenotype but also fostered the production of cartilage specific extracellular matrix. To further improve the biological activities of the HA-c-HGP gels, bone morphogenetic protein 2 (BMP-2) was loaded into the immobilized HGPs. BMP-2 was released from the HA-c-HGP gels in a controlled manner with reduced initial burst over prolonged periods of time. The HA-c-HGP gels are promising candidates for use as bioactive matrices for cartilage tissue engineering.

Regulation of Stem Cell Function in an Engineered Vocal Fold-Mimetic Environment.

Human mesenchymal stem cells (hMSCs) have been proposed as therapeutic cells for the treatment of vocal fold (VF) scarring. Although functional recovery was observed in animal models after stem cell injection, it is not clear how injected stem cells interact locally with the extracellular matrix (ECM) of the lamina propria (LP) and how such interactions affect stem cell behaviors to improve function. Herein, we developed an in vitro cell culture platform where hMSCs were encapsulated in a LP-mimetic matrix, derived from hyaluronic acid (HA), poly(ethylene glycol) (PEG) and collagen, and cultured dynamically in a custom-designed VF bioreactor. The cell culture system was characterized by oscillatory shear rheology, laser doppler vibrometry (LDV), and digital image correlation (DIC). A constitutive finite element analysis (FEA) model was further developed to predict vibratory responses of the hydrogel. LDV analysis demonstrated an average displacement of 47 µm in the center of the hydrogel construct at 200 Hz applied frequency without any harmonics. The predicted strains throughout the hydrogel ranged from 0 to 0.03, in good agreement with reported values for the VF. The 3D cellular construct was subjected to vibrational stimulations at 200 Hz for an optimized duration of 1 h, as confirmed by a maximal c-Fos upregulation at the transcript level. Vibrational culture over a 3-day period with a 1h-on/1h-off pattern did not compromise the overall cell viability, but resulted in a significant downregulation of fibrogenic markers and diminished staining for alpha smooth muscle actin (αSMA). Collectively, high frequency mechanical loading resulted in the loss of myofibrogenic potential and a shift away from a fibrotic phenotype.

P2X7 nucleotide receptor plays an important role in callus remodeling during fracture repair.

The P2X7 nucleotide receptor (P2X7R) is an ATP-gated ion channel expressed in bone cells. Homozygous null P2X7R (P2X7R(-/-)) mice have reduced bone formation, so we hypothesized that P2X7R(-/-) mice have impaired fracture healing compared to P2X7R(+/+) control mice. To test the hypothesis, adult P2X7R(-/-) mice and P2X7R(+/+) mice were studied. Osteotomy of the right femur was performed and a stainless-steel pin was inserted into the medullary cavity to stabilize the fracture site. No differences in callus development were seen in the radiograph, micro computed tomography, or dual-energy x-ray absorptiometry measurements. Mechanical testing showed that the recovery of ultimate force, stiffness, and energy to failure were slightly decreased in P2X7R(-/-) mice compared with the control. Histomorphometric measurements of the callus revealed that mineralizing surface and bone formation were significantly decreased, by 22% (p < 0.001) and 29% (p < 0.05), respectively, in P2X7R(-/-) mice in comparison with the wild-type control. These data show that a null mutation of the P2X7R does not affect the amount of callus formed in our osteotomy fracture model. However, callus remodeling was significantly delayed. Our data suggest the different role of the P2X7R in woven bone and lamellar bone formation.

Culture of Mesenchymal Stem Cells in a Hydrogel Model of Vocal Fold Lamina Propria.

Stem cell injection has been proposed as an alternative approach for the restoration of vocal fold (VF) function in patients with VF scarring. To assess the therapeutic efficacy of this treatment strategy, we evaluated the behaviors of human mesenchymal stem cells (hMSCs) in hydrogels derived from thiolated hyaluronic acid (HA-SH) and poly(ethylene glycol) diacrylate (PEG-DA) entrapping assembled collagen fibrils (abbreviated as HPC gels). Three hydrogel formulations with varying amounts of collagen (0, 1 and 2 mg/mL) but a fixed HA-SH (5 mg/mL) and PEG-DA (2 mg/mL) concentration, designated as HPC0, HPC1 and HPC2, were investigated. The HPC gels exhibit similar pore sizes (35-50 nm) and AFM indentation moduli (~175 Pa), although the elastic shear modulus for HPC1 (~32 Pa) is lower than HPC0 and HPC2 (~55 Pa). Although HPC1 and HPC2 gels both promoted the development of an elongated cell morphology, greater cell spreading was observed in HPC2 than in HPC1 by day 7. At the transcript level, cells cultured in HPC1 and HPC2 gels had an increased expression of fibronectin and integrin ß1, but a decreased expression of tissue inhibitor of metalloproteinase-1, collagen types I/III and HA synthase-1 when compared to cells cultured in HPC0 gels. Cellular expression of connective tissue growth factor was also elevated in HPC1 and HPC2 cultures. Importantly, the HPC2 hydrogels promoted a signficant up-regulation of matrix metalloproteinase 1, transforming growth factor ß1, and epithelial growth factor receptor, indicating an increased tissue turnover. Overall, hMSCs cultured in HPC2 gels adopt a phenotype reminiscent of cells involved in the wound healing process, providing a platform to study the effectiveness of therapeutic stem cell treatments for VF scarring.

Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca(2+)- and ATP-dependent in MC3T3-E1 osteoblasts.

To determine the role of Ca2+ signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca2+ signaling during application of fluid shear stress (FSS). FSS only activated ERK1/2, rapidly inducing phosphorylation within 5 min of the onset of shear. Phosphorylation of ERK1/2 (pERK1/2) was significantly reduced when Ca2+i was chelated with BAPTA or when Ca2+ was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca2+ channel and the mechanosensitive cation-selective channel blocked FSS-induced pERK1/2. Inhibition of phospholipase C with U73122 significantly reduced pERK1/2. This inhibition did not result from blockage of intracellular Ca2+ release, but a loss of PKC activation. Recent data suggests a role of ATP release and purinergic receptor activation in mechanotransduction. Apyrase-mediated hydrolysis of extracellular ATP completely blocked FSS-induced phosphorylation of ERK1/2, while the addition of exogenous ATP to static cells mimicked the effects of FSS on pERK1/2. Two P2 receptors, P2Y2 and P2X7, have been associated with the anabolic responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X7 knockout mice, we found that the P2X7, but not the P2Y2, purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca2+-dependent ATP release, however both increased Ca2+i and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X7, but not P2Y2, purinergic receptors.

Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3.

Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone densityover time. It affects one in two women and one in four men, age 50 and older. New treatmentsthat specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, thatacts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesisin-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cellsremains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by sizeexclusion chromatography and verified the conjugation and stability using UV/VIS and Fouriertransform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s andthe conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biologicalresponse similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealedthat CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacologicalinhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized bycaveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cellthrough caveolae mediated endocytosis.

Synthetic Peptide CK2.3 Enhances Bone Mineral Density in Senile Mice.

BACKGROUND: Osteoporosis is a silent disease caused by low bone mineral density that results in bone fractures in 1 out of 2 women and 1 in 4 men over the age of 50. Although several treatments for osteopenia and osteoporosis are available, they have severe side effects and new treatments are desperately needed. Current treatments usually target osteoclasts and inhibit their activity or differentiation. Treatments that decrease osteoclast differentiation and activity but enhance osteogenesis and osteoblast activity are not available. We recently developed a peptide, CK2.3, that induces bone formation and increases bone mineral density as demonstrated by injection over the calvaria of 6 to 9-day-old mice and tail vein injection of 8-week-old mice. CK2.3 also decreased osteoclast formation and activity. However, these studies raise questions: does CK2.3 induce similar results in old mice and if so, what is the effective CK2.3 concentration and, is the bone mineral density of vertebrae of the spinal column increased as well? METHODS: CK2.3 was systematically injected into the tail vein of female 6-month old mice with various concentrations of CK2.3: 0.76 µg/kg, 2.3 µg/kg, or 6.9 µg/kg per mice. Mice were sacrificed one week, two weeks, and four weeks after the first injection. Their spines and femurs were collected and analyzed for bone formation. RESULTS: Femur and lumbar spine analyses found increased bone mineral density (BMD) and mineral apposition rate, with greater stiffness observed in femoral samples four weeks after the first injection. Histochemistry showed that osteoclastogenesis was suppressed in CK2.3 treated senile mice. CONCLUSIONS: For the first time, this study showed the increase of lumbar spine BMD by CK2.3. Moreover, it showed that enhancement of femur BMD was accompanied by increased femur stiffness only at medium concentration of CK2.3 four weeks after the first injection indicating the maintenance of bone's structural integrity by CK2.3.

PTH-induced actin depolymerization increases mechanosensitive channel activity to enhance mechanically stimulated Ca2+ signaling in osteoblasts.

UNLABELLED: Disruption of the actin cytoskeleton with cytochalasin D enhanced the mechanically induced increase in intracellular Ca(2+) ([Ca(2+)](i)) in osteoblasts in a manner similar to that of PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Patch-clamp analyses show that the MSCC is directly influenced by alterations in actin integrity. INTRODUCTION: PTH significantly enhances the fluid shear-induced increase in [Ca(2+)](i) in osteoblasts, in part, through increased activation of both the mechanosensitive, cation-selective channel (MSCC) and L-type voltage-sensitive Ca(2+) channel (L-VSCC). Both stimuli have been shown to produce dynamic changes in the organization of the actin cytoskeleton. In this study, we examined the effects of alterations in actin polymerization on [Ca(2+)](i) and MSCC activity in MC3T3-E1 and UMR-106.01 osteoblasts in response to shear +/- PTH pretreatment. MATERIALS AND METHODS: MC3T3-E1 or UMR-106.01 cells were plated onto type I collagen-coated quartz slides, allowed to proliferate to 60% confluency, and mounted on a modified parallel plate chamber and subjected to 12 dynes/cm(2). For patch-clamp studies, cells were plated on collagen-coated glass coverslips, mounted on the patch chamber, and subjected to pipette suction. Modulators of actin cytoskeleton polymerization were added 30 minutes before the experiments, whereas channel inhibitors were added 10 minutes before mechanical stimulation. All drugs were maintained in the flow medium for the duration of the experiment. RESULTS AND CONCLUSIONS: Depolymerization of actin with 1-5 microM cytochalasin D (cyto D) augmented the peak [Ca(2+)](i) response and increased the number of cells responding to shear, similar to the increased responses induced by pretreatment with 50 nM PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Inhibition of the MSCC with Gd(3+) significantly blocked both the peak Ca(2+) response and the number of cells responding to shear in cells pretreated with either PTH or cyto D. Inhibition of the L-VSCC reduced the peak [Ca(2+)](i) response to shear in cells pretreated with PTH, but not with cyto D. Patch-clamp analyses found that addition of PTH or cyto D significantly increased the MSCC open probability in response to mechanical stimulation, whereas phalloidin significantly attenuated the PTH-enhanced MSCC activation. These data indicate that actin reorganization increases MSCC activity in a manner similar to PTH and may be one mechanism through which PTH may reduce the mechanical threshold of osteoblasts.