Role of the low‐density lipoprotein receptor‐related protein‐1 in regulation of chondrocyte differentiation

The low‐density lipoprotein receptor‐related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/β‐catenin (βcat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/β–catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3β, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) ζ was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCζ was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1. J. Cell. Physiol. 222:138–148, 2010. © 2009 Wiley‐Liss, Inc.     

Swelling and flow properties of tubular poly(vinyl alcohol) gels

Swelling and flow properties of tubular poly(vinyl alcohol) (PVA) hydrogels prepared with the cooling method were investigated using an inflation testing method. When the tubular hydrogel in liquid paraffin was inflated by using liquid paraffin as a pressure transmitting medium, namely in the case that the liquids inside and outside the gel are both liquid paraffin (P/P combination), the gel showed a slight volume change determined by Poisson's ratio of the gel. When the gel in water was inflated by liquid paraffin (P/W combination), the gel swelled to large extent compared with the case of P/P. The hydrogel in W/W combination, namely in the situation that the gel was immersed in water and also inflated by water, showed a very large volume change if the comparison was done at the same pressure. The origin of the volume change in P/P, P/W and W/W combinations is discussed. The volume change in P/P was governed by the Poisson ratio as a material constant (mu 0) of the PVA gels, and the gels swelled by the change in the application of pressure (or deformation) in P/W. The volume change in W/W was closely related to the flow of solvent in the gel.     

Activation of epidermal growth factor receptor promotes late terminal differentiation of cell-matrix interaction-disrupted keratinocytes

The biological effects of epidermal growth factor receptor (EGFR) activation may differ between epidermal suprabasal and basal keratinocytes, since growth factors are mitogenic in adherent cells only in the presence of cell-extracellular matrix (ECM) interaction. To investigate biological effects of EGFR activation on keratinocytes without cell-ECM interaction, we cultured normal human keratinocytes on polyhydroxyethylmethacrylate-coated plates, which disrupt cell-ECM but not cell-cell interaction. The cells initially expressed keratin 10 (K10) and then profilaggrin, mimicking sequential differentiation of epidermal suprabasal keratinocytes. The addition of EGF or transforming growth factor-alpha promoted late terminal differentiation (profilaggrin expression, type 1 transglutaminase expression and activity, and cornified envelope formation) of the suspended keratinocytes, while suppressing K10 expression, an early differentiation marker. These effects were attenuated by EGFR tyrosine kinase inhibitor PD153035 or an anti-EGFR monoclonal antibody, whereas protein kinase C inhibitors H7 and bisindolylmaleimide I or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 abolished profilaggrin up-regulation but not K10 suppression. Since the antidifferentiative role of EGFR on cell-ECM interaction-conserved keratinocytes has been well documented, our results indicate that the biological effects of EGFR on keratinocytes are influenced by cell-ECM interaction and suggest that EGFR activation promotes rather than inhibits the terminal differentiation of suprabasal epidermal keratinocytes.     

Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein. Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF. Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis. These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix. High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.     

Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.     

Characterization of drug-specific T cells in phenobarbital-induced eruption

Phenobarbital has a high potential to elicit adverse reactions including severe skin eruptions and systemic involvements among the worldwide-prescribed drugs. Although phenobarbital hypersensitivity is thought to be mediated by T cells specific to the drug, its precise mechanism remains not fully elucidated. To characterize T cells reactive with phenobarbital, we generated drug-specific T cell clones and lines from PBMCs of patients with phenobarbital hypersensitivity showing various degrees of cutaneous and extracutaneous involvements. Although the TCR Vbeta repertoire and phenotype in the T cell clones/T cell lines were heterogeneous among the patients, Vbeta13.1(+) and Vbeta5.1(+) clones or lines were raised from the individuals examined who possessed different HLA haplotypes. Histopathological examination suggested that Vbeta5.1(+)CD8(+) T cells and Vbeta13.1(+) T cells played a role in cutaneous and extracutaneous involvements, respectively. A Vbeta13.1(+)CD4(+) clone was found to proliferate in response to the Ag with processing-impaired, fixed APCs. Most of the clones and lines belonged to the Th2 phenotype, producing IL-4 and IL-5 but not IFN-gamma upon phenobarbital stimulation. Clones/lines with Th1 or Th0 phenotypes also constituted minor populations. These observations clearly indicate the heterogeneity and a marked individual deviation of reactive T cell subsets among the patients in terms of CD4/8 phenotype, Vbeta repertoire, Ag recognition pattern, and cytokine production; and thus provide evidence whereby each pathogenic T cell subset contributes to special elements of clinical presentation.     

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade.

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.     

Expression of osteopontin in Meckel’s cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis

 The localization of osteopontin (OP) was examined in Meckel’s cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix. Accepted: 26 May 1998