Chondrocytic cells express the taurine transporter on their plasma membrane and regulate its expression under anisotonic conditions.

Taurine is a small organic osmolyte which participates in cell volume regulation. Chondrocytes have been shown to accumulate and release taurine; in bone, taurine participates in bone metabolism. However, its role in skeletal cells is poorly understood, especially in chondrocytes. This study investigated the regulation of taurine transporter in chondrocytic cells. We examined the transcriptional regulation of the taurine transporter under anisotonia by reporter gene and real-time RT-PCR assays. The effect of providing supplementary taurine on cell viability was evaluated with the lactate dehydrogenase release assay. The localization of the taurine transporter in human chondrosarcoma cells was studied by overexpressing a taurine transporter-enhanced green fluorescent protein. We observed that the transcription of the taurine transporter gene was up-regulated in hypertonic conditions. Hyperosmolarity-related cell death could be partly abolished by taurine supplementation in the medium. As expected, the fluorescently labeled taurine transporter localized at the plasma membrane. In polarized epithelial MDCK cells, the strongest fluorescence signal was located in the lateral cell membrane area. We also observed that the taurine transporter gene was expressed in several human tissues and malignant cell lines. This is the first study to present information on the transcriptional regulation of taurine transporter gene and the localization of the taurine transporter protein in chondrocytic cells.

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium.

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell-cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell-cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell-cell and cell-ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.

Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells.

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.

Genome-wide microarray analysis of MG-63 osteoblastic cells exposed to ultrasound.

It is well documented that low intensity pulsed ultrasound can be clinically used to accelerate bone fracture healing. Additionally, in vitro studies have shown that ultrasound can, for instance, increase mineralization, collagen production and alkaline phosphatase activity in osteoblasts. Despite the extensive research on the subject, the exact mechanism of ultrasound effect on bone cell gene regulation has not yet been deduced. In this study, we made an effort to reveal the features of genome-wide transcriptional response of osteoblast-type cells to ultrasound. MG-63 osteoblastic cell transcriptome was analyzed with whole genome microarray either 6 or 24 h after 30 min long exposure to 1.035 MHz pulsed ultrasound with three different acoustic pressures. Special attention was paid to the experimental design to minimize thermal effects and unwanted reflections of ultrasound. Microarray analysis suggested that ultrasound affects the genes involved with cellular membranes, and regulation of transcription as well. Several plasma membrane solute carriers were also regulated by ultrasound. It also changed the transcript level of several transcription factors belonging to the zinc finger proteins. However, ultrasound did not clearly promote genes involved with osteoblast differentiation.

The lack of effect of glucosamine sulphate on aggrecan mRNA expression and (35)S-sulphate incorporation in bovine primary chondrocytes.

Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and (35)S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 microM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited (35)S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or (35)S-sulphate incorporation.

Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes.

Mechanical forces can stimulate the production of extracellular matrix molecules. We tested the efficacy of ultrasound to increase proteoglycan synthesis in bovine primary chondrocytes. The ultrasound-induced temperature rise was measured and its contribution to the synthesis was investigated using bare heat stimulus. Chondrocytes from five cellular isolations were exposed in triplicate to ultrasound (1 MHz, duty cycle 20%, pulse repetition frequency 1 kHz) at average intensity of 580 mW/cm2 for 10 minutes daily for 1-5 days. Temperature evolution was recorded during the sonication and corresponding temperature history was created using a controllable water bath. This exposure profile was used in 10-minute-long heat treatments of chondrocytes. Heat shock protein 70 (Hsp70) levels after one-time treatment to ultrasound and heat was analyzed by Western blotting, and proteoglycan synthesis was evaluated by 35S-sulfate incorporation. Ultrasound treatment did not induce Hsp70, while heat treatment caused a slight heat stress response. Proteoglycan synthesis was increased approximately 2-fold after 3-4 daily ultrasound stimulations, and remained at that level until day 5 in responsive cell isolates. However, chondrocytes from one donor cell isolation out of five remained non-responsive. Heat treatment alone did not increase proteoglycan synthesis. In conclusion, our study confirms that pulsed ultrasound stimulation can induce proteoglycan synthesis in chondrocytes.

cDNA array reveals mechanosensitive genes in chondrocytic cells under hydrostatic pressure.

Hydrostatic pressure (HP) has a profound effect on cartilage metabolism in normal and pathological conditions, especially in weight-bearing areas of the skeletal system. As an important component of overall load, HP has been shown to affect the synthetic capacity and well-being of chondrocytes, depending on the mode, duration and magnitude of pressure. In this study we examined the effect of continuous HP on the gene expression profile of a chondrocytic cell line (HCS-2/8) using a cDNA array containing 588 well-characterized human genes under tight transcriptional control. A total of 51 affected genes were identified, many of them not previously associated with mechanical stimuli. Among the significantly up-regulated genes were immediate-early genes, and genes involved in heat-shock response (hsp70, hsp40, hsp27), and in growth arrest (GADD45, GADD153, p21(Cip1/Waf1), tob). Markedly down-regulated genes included members of the Id family genes (dominant negative regulators of basic helix-loop-helix transcription factors), and cytoplasmic dynein light chain and apoptosis-related gene NIP3. These alterations in the expression profile induce a transient heat-shock gene response and activation of genes involved in growth arrest and cellular adaptation and/or differentiation.

Geldanamycin activates Hsp70 response and attenuates okadaic acid-induced cytotoxicity in human retinal pigment epithelial cells.

Reversible protein phosphorylation regulates the biological activities of many human proteins involved in crucial cellular processes, e.g., protein-protein interactions, cell signaling, gene transcription, cell growth, and death. A malfunction of cellular homeostasis in retinal pigment epithelial (RPE) cells is involved in the age-related retinal degeneration. In this study, we examined cytotoxicity in human RPE cells subjected to the protein phosphatase inhibitor, okadaic acid (OA). Moreover, the influence of Hsp90 inhibitor geldanamycin (GA), a benzoquinone ansamycin, in cytoprotection was assessed. Hsp70 protein levels were analyzed by Western blot. Cellular viability was determined by LDH and MTT assays. To study apoptotic cell death, caspase-3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate and Hoechst dye was used to visualize nuclear morphology. OA treatment caused morphological changes and induced cytotoxicity by caspase-3-independent manner in the RPE cells. No evidence of nuclear fragmentation was observed in response to OA. Interestingly, GA treatment accumulated Hsp70 protein and attenuated OA-induced cytotoxicity. This study suggests that Hsp70 and Hsp90 are closely related to cytoprotection of RPE cells in response to protein phosphatase inhibition.

Geldanamycin increases 4-hydroxynonenal (HNE)-induced cell death in human retinal pigment epithelial cells.

Development of age-related macular degeneration (AMD) is associated with functional abnormalities and cell death in retinal pigment epithelial (RPE) cells attributable to oxidative stress. To minimize the adverse effects of oxidative stress, cells activate their defence systems, e.g., via increased expression of heat shock protein (Hsp), activation of stress sensitive AP-1 and NF-kappaB transcription factors. In this study, we examined the accumulation of Hsp70 protein, activation of AP-1 and NF-kappaB transcription factors in human ARPE-19 cells subjected to a 4-hydroxynonenal (HNE)-induced oxidative stress. In addition, the influence of Hsp90 inhibitor geldanamycin (GA) was studied in HNE-treated cells. Mitochondrial metabolic activity and apoptosis were determined to evaluate cell death in the ARPE-19 cells. The ARPE-19 cells showed increased accumulation of Hsp70 protein before of the cytotoxic hallmarks appearing in response to HNE. In contrast, increased DNA-binding activities of AP-1 or NF-kappaB transcription factors were not seen under HNE insults. Interestingly, GA significantly increased cell death in the HNE-treated cells, which was involved in caspase-3 independent apoptosis. This study reveals that the Hsps have an important role in the cytoprotection of RPE cells subjected to HNE-derived oxidative stress.

Reticulon 4 in chondrocytic cells: barosensitivity and intracellular localization.

Members of the reticulon gene family are endoplasmic reticulum (ER)-related proteins expressed in various human tissues, but their molecular functions are not understood. The reticulon 4 subfamily consists of three members, reticulon 4/Nogo-A, -B and -C. Reticulon 4-A is under intense investigation because of its inhibitory effect on neurite outgrowth, and reticulon 4-B has been suggested to induce apoptosis. Reticulon 4-C, the shortest member of this subfamily, is the least characterized. Reticulons are presumably guided to endoplasmic reticulum by a putative N-terminal retention motif. In this study the expressions of reticulon 4 subtypes in human chondrosarcoma cell line and in primary bovine chondrocytes were analyzed on mRNA level. These cell types, exposed to strong mechanical forces in vivo, were subjected to high hydrostatic pressure and mechanical stretch to study the possible mechanosensitivity of reticulon 4 genes. In addition, a green fluorescent protein-tagged reticulon 4-C and a fusion protein with mutated endoplasmic reticulum retention signal were used to study the significance of the C-terminal translocation signal (the di-lysine motif). As the result, both cell types expressed the three main isoforms of reticulon 4 family. The steady-state level of reticulon 4-B mRNA was shown to be up-regulated by pressure, but not by mechanical stretch indicating transcriptional barosensitivity. The reticular distribution pattern of reticulon 4-C was observed indicating a close association with endoplasmic reticulum. Interestingly, this pattern was maintained despite of the disruption of the putative localization signal. This suggests the presence of another, yet unidentified endoplasmic reticulum retention mechanism.

Hydrostatic pressure-induced changes in cellular protein synthesis.

Hydrostatic pressure is a well-known effector of cellular protein synthesis. High continuous hydrostatic pressure inhibits protein synthesis in general. It has been known for a long time that 30S ribosomal subunit is associated with the effects of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood. Our new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic pressure. Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells. The presence of pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells. Accumulation of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis, although this response appears clearly weaker than during heat stress.

Stress responses of mammalian cells to high hydrostatic pressure.

High hydrostatic pressure causes stress response in many types of mammalian cells. We have previously shown that an accumulation of heat shock protein 70 (Hsp70) in a chondrocytic cell line occurred without an activation of the gene itself. Stabilization of the hsp70 mRNA was shown to be the reason for the Hsp70 stress response in the pressurized cells. Since accumulation of Hsp70 in pressurized cells indicated that high hydrostatic pressure induces a stress response without heat shock transcription factor activation, we decided to investigate the activation of two other stress-associated transcription factors, activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB). Induction of Hsp70 in immortalized and primary chondrocytes, murine Neuro-2a neuroblastoma and HeLa cervical carcinoma cell lines was investigated at both mRNA and protein levels. In immortalized chondrocytes and HeLa cells, hsp70 mRNA levels were clearly elevated after 6 hours of the onset of 30 MPa continuous hydrostatic pressure, while in primary chondrocytes and Neuro-2a cells (the cells known to be stress-sensitive) no induction was observed. Surprisingly, neither heat shock nor high hydrostatic pressure could induce the hsp70 mRNA in Neuro-2a cells, although an activation of heat shock transcription factor could be observed in heat-shocked cells. No activation of the AP-1 and NF-kappaB binding to their target DNA sequences could be shown in the immortalized chondrocytes.

Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study.

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha6, and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alphaE and beta8, and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.

Specific induction of heat shock protein 90beta by high hydrostatic pressure.

In chondrocytes, a low-amplitude intermittent hydrostatic pressure induces production of extracellular matrix molecules, while high hydrostatic pressure inhibits it. High pressure increases cellular heat shock protein 70 level in a number of cell types on account of increased stabilisation of the heat shock protein 70 mRNA. In our experiments, only bovine primary chondrocytes, but not an immortalized chondrocytic cell line, could resist the induction of the stress response in the presence of continuous 30 MPa hydrostatic pressure. We have recently shown that protein synthesis is required for the stabilization. According to two-dimensional gel electrophoresis the synthesis of heat shock protein 90 was also increased in a chondrocytic cell line and in HeLa cells, and mass spectrometric analysis suggested that the induction was rather due to increase in heat shock protein 90beta than in heat shock protein 90alpha. The stress response was rather intense in HeLa cells, therefore, we investigated the effect of continuous 30 MPa hydrostatic pressure on the expression of the two heat shock protein 90 genes in HeLa cells using Northern and Western blot analyses. Heat shock protein 90beta mRNA level increased within 6 hours of exposure to 30 MPa hydrostatic pressure, while hsp90alpha level remained stable. At protein level there was a clear increase in the heat shock protein 90beta/heat shock protein 90alpha ratio, too. These results show a specific regulation of stress proteins in cells exposed to high hydrostatic pressure.

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis.

Hydrostatic pressure has a profound effect on cartilage tissue and chondrocyte metabolism. Depending on the type and magnitude of pressure various responses can occur in the cells. The mechanisms of mechanotransduction at cellular level and the events leading to specific changes in gene expression are still poorly understood. We have previously shown that induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA. In this study, our aim was to examine the effect of high pressure on gene expression profile and to study whether stabilization of mRNA molecules is a general phenomenon under this condition. For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin, fibronectin, and collagen types VI and XVI mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon.

High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2.

High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.