Kappei Kobayashi

Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture.

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs...

Kinetics of Cytokine Production in the Cornea and Trigeminal Ganglion of C57BL/6 Mice after Corneal HSV-1 Infection

To investigate the role of cytokines in the pathogenesis of acute herpetic keratitis (HK), we examined the kinetics of cytokine expression in the corneas and the trigeminal ganglia (TG) of C57BL/6Cr (B6) mice after herpes simplex virus type 1 (HSV-1) infection and observed the influence of the targeted disruption of interferon-gamma (IFN-gamma) gene on the clinical course of HK and/or viral clearance. Following corneal infection with HSV-1 Amakata strain, all corneas developed a typical dendritic keratitis. Quantitative analysis using enzyme-linked immunosorbent assay (ELISA) revealed that the expression of interleukin-1alpha (IL-1alpha), IL-5, IL-6, and IFN-gamma in corneas and TGs significantly elevated immediately after infection, peaked between days 2 and 7 postinfection (p.i.), and then diminished. One exception was IFN-gamma, whose expression significantly persisted in the TGs until day 30 p.i. An additional experiment using IFN-gamma-/- (gko) mice revealed that there was no significant difference in the peak level of viral replication in corneas and TGs between gko and B6 mice, although gko mice showed a significant delay of virus clearance in both corneas and TGs (p < 0.005) and higher mortality rate than B6 mice after HSV-1 infection (p < 0.01). These data suggest that the production of proinflammatory cytokines closely correlates with the pathogenesis of HK, and that IFN-gamma plays an important role in enhancing viral clearance from the cornea and TG.

Analysis of heat shock proteins and cytokines expressed during early stages of osteoarthritis in a mouse model

OBJECTIVE Osteoarthritis (OA) is a debilitating disease of the joints. The joints of affected individuals are characterized by a progressive degeneration of articular cartilage leading to inflammation and pain. The expression of heat shock proteins (HSPs) is a ubiquitous self-protective mechanism of all cells under stress, furthermore, the synovium of osteoarthritic individuals contains high levels of cytokines. This study seeks to establish the role of HSPs and cytokines in OA. METHODS We have investigated the presence of HSPs and cytokines in articular cartilage during early stages of OA in a mouse that is known to develop spontaneous OA lesions (C57 black mouse). The articular cartilage from closely related mice (C57BL/6) was used as control. Messenger RNAs (mRNAs) for HSPs (HSP32, HSP47, HSP60, HSP70, HSP84 and HSP86) and cytokines [interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS The mRNA levels of HSP47, HSP70, HSP86, IL-6, and IFN-gamma were up-regulated in the cartilage of C57 black mice, whereas, the level of expression of HSP32, HSP60, HSP84 and IL-1 beta remained unchanged. Furthermore, the expression of IL-1 beta, IL-6, TNF-alpha and IFN-gamma mRNA was associated with expression of HSP60, HSP47, HSP70 and HSP70/HSP86 mRNA, respectively. CONCLUSIONS The findings in this study suggest that chondrocytes are conditioned under non-physiological stress during early stages of OA, In addition, among HSPs, HSP70 was associated with two different highly expressed cytokines in C57 black mice, indicating the possible role of HSP70 as a characteristic indicator of early stage of OA.

Herpes Simplex Virus‐Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post‐infection. HSP70 synthesis and accumulation increased in HSV‐infected cells. Irradiation of HSV with UV‐light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.

Messenger RNA expression of heat shock proteins (HSPs) during ocular development.

The heat shock proteins (HSPs) are believed to act as molecular chaperones which appear to play some roles in regulation of normal protein folding and also in preventing damage to protein structures under various conditions of environmental stress. We examined the expression of the major HSP families, HSP60, 70 and 90 families and small HSP32, at the mRNA level in ocular development. Expression of HSP32, HSP60, HSP70, HSP84, HSP86 and heat shock cognate protein (HSC)70 mRNAs was examined by in situ hybridization. HSC70, HSP84, HSP86 and HSP60 mRNAs were expressed strongly in all ocular tissues during early stages 3 to 5, corresponding to embryonic day (E)11.5 to E14.5. At stages 6 to 7 (E15.5 to E18.5), the expression of these four mRNA species was decreased markedly in most ocular tissue, while in the retina strong HSC70 and HSP86 mRNA expression was still detected. HSP32 and HSP70 mRNAs were not detected at any stage. These results suggest that the expression of HSPs is developmentally regulated through ocular organogenesis, and the proteins may play some important roles in ocular development.

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.

Induced Expression and Localization to Nuclear-Inclusion Bodies of hsp70 in Varicella-Zoster Virus-Infected Human Diploid Fibroblasts

The expression and subcellular localization of cellular heat‐shock protein hsp70 were examined in varicella‐zoster virus (VZV)‐infected human diploid fibroblasts. Infection with VZV elevated the steady‐state levels of hsp70 mRNA by 24 hr post‐infection (hpi). Western blotting analysis revealed an increase in accumulation of hsp70 from 24 hpi. Subcellular localization of the hsp70 in VZV‐infected cells was examined by indirect immunofluorescence. In most VZV‐infected cells, hsp70 was localized to inclusion bodies induced in the cell nucleus by infection with VZV. In some cells, however, the remaining parts of the cell nucleus and the cytoplasm were also stained with anti‐hsp70 antibody. These results indicate that infection with VZV induces the expression of hsp70 and its localization to VZV‐specific inclusion bodies, which suggests the involvement of hsp70 in molecular events within inclusion bodies.

Gene Delivery to Chondrocytes Using Adenovirus Vector

The objective of this study was to investigate the effects of adenovirus vector (Ax-)mediated gene transduction of E. coli β-galactosidase (LacZ) and transforming growth factor-β1 (TGF-β1) into a human chondrocyte-like cell line (HCS-2/8). The expression of transduced genes and their expression periods were examined by 5-bromo-4-chloroindolyl-β-D-galactoside (X-gal) staining, Northern blotting, ELISA, and Western blotting. To assess the influence of TGF-β1 gene transduction, the expression of mRNAs of type II collagen, proteoglycan core protein, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were examined by Northern blotting. Staining with X-gal indicated that the genes were transduced into 99% of the cells. Expression of the transduced genes in the cells was continued for at least 21 days. Transduction of the TGF-β1 gene enhanced mRNA expressions of type II collagen and proteoglycan core protein, but suppressed MMP-3 mRNA expression in the cells. These results indicate Ax is useful in chondrocyte gene therapy, and it could be an efficient mediator of TGF-β1 gene transduction.