Tomoo Tsukazaki

Localisation of apoptosis and expression of apoptosis related proteins in the synovium of patients with rheumatoid arthritis.

OBJECTIVES: To investigate whether apoptosis occurs in the synovium of rheumatoid arthritis (RA), and the intermediate molecules operating in this process. METHODS: DNA fragmentation was detected by in situ nick end labelling (ISNEL) in the synovium of patients with RA (n = 11) and control patients with femoral neck fracture (n = 5). The expression of proteins p53, p21WAFI/CIPI, c-myc, proliferating cell nuclear antigen (PCNA), and Bcl-2 was also examined by immunohistochemistry. RESULTS: ISNEL positive synovial cells with apoptosis specific morphology were detected in extremely limited areas in only two RA synovial tissue specimens. Proteins p53, p21WAFI/CIPI, and c-myc, known inducers of apoptosis or cell cycle arrest or both, were expressed in the sublining cells independent of ISNEL positive cells. PCNA, a marker for cell proliferation, was observed in the synovial lining cells. Bcl-2, an inhibitor of apoptosis, was expressed mainly in infiltrated lymphocytes and in parts of the sublining layer cells of RA; it also did not correspond with ISNEL staining. CONCLUSIONS: Our findings indicate that RA synovial cells undergo apoptosis in addition to cell proliferation, but the frequency of apoptosis was very low. We suspect that the apoptotic process in the RA synovium may be suppressed by over-expression of Bcl-2. Although expressed proteins p53, p21WAFI/CIPI, and c-myc were present in the RA synovium, these protooncogenes are probably not implicated in the apoptotic process.

Norepinephrine Enhances Fibrosis Mediated by TGF-β in Cardiac Fibroblasts

Cardiac fibrosis results from proliferation of interstitial fibroblasts and concomitant increased biosynthesis of extracellular matrix (ECM) components and is often complicated by cardiac hypertrophy. This study was conducted to investigate whether norepinephrine (NE) potentiates transforming growth factor-&bgr; (TGF-&bgr;)–induced cardiac fibrosis. The expression of the cardiac ECM proteins, plasminogen activator inhibitor-1 (PAI-1), fibronectin, and collagen type I, was examined by Western blotting using extracts from neonatal rat primary cardiac fibroblasts. In cardiac fibroblasts, treatment with a combination of NE and TGF-&bgr;1 increased cell proliferation and ECM expression. Luciferase assays were conducted to clarify the effect of NE on TGF-&bgr; signaling. TGF-&bgr;1 (1 ng/mL) increased the specific signaling activity 2-fold, whereas the combination of NE (10 &mgr;mol/L) and TGF-&bgr;1 (1 ng/mL) resulted in an approximate 10-fold increase in specific signaling activity. We confirmed that treatment with NE markedly enhances TGF-&bgr;–induced phosphorylation of activating transcription factor 2 (ATF-2). These results indicated that NE has a synergistic effect on TGF-&bgr; signaling. To determine whether this activation by NE was mediated by the TGF-&bgr;1 receptor, we used a dominant negative vector of the TGF-&bgr;1 type II receptor, and the synergistic effects were inhibited. Furthermore, this synergistic effect was attenuated by a specific inhibitor of p38, SB203680. These data indicate that NE enhances cardiac fibrosis through TGF-&bgr;1 post-receptor signaling, predominantly via the p38 MAP kinase pathway.

Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines.

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD‐C6, from Runx2‐deficient mouse embryos. RD‐C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) or transduction of BMP‐2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD‐C6 cells. BMP‐2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD‐C6 cells with or without rhBMP‐2 treatment demonstrated that BMP‐2 upregulated 66 genes including 13 transcription‐related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD‐C6 cells, we transplanted RD‐C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP‐2. RD‐C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP‐2 induces Runx2‐deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2‐independent pathway, but it failed to induce these cells to differentiate into bone‐forming osteoblasts and mature chondrocytes. J. Cell. Physiol. 211: 728–735, 2007.

PKC δ mediates ionizing radiation-induced activation of c-Jun NH2-terminal kinase through MKK7 in human thyroid cells

The thyroid gland is one of the most sensitive organs in ionizing radiation (IR)-induced carcinogenesis. To determine, therefore, the specific cascade of IR-induced signal transduction in human thyroid cells, we investigated the functional role of protein kinase C (PKC), especially its interlocking activation of c-Jun NH2-terminal kinase (JNK) pathway. In the present study, using adenovirus expression vectors for diverse dominant-negative (DN) types of PKC isoforms (α, β2, δ, ε and ζ) expressed in primary cultured human thyroid cells, only DN/PKC δ suppressed IR-induced JNK activation. In addition, Rottlerin, a PKC δ specific inhibitor, inhibited IR-induced JNK activation. IR-induced activation of transcription factor AP-1, downstream target of JNK, was also attenuated by DN/PKC δ. To examine the involvement of upstream kinases of JNK, we performed immune-complex kinase assays of mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. IR activated MKK7 but not MKK4, and this activation was inhibited by Rottlerin. Furthermore, IR-induced JNK activation was suppressed by overexpression of kinase-deficient MKK7. Our results indicate that IR selectively activates the cascade of PKC δ-MKK7-JNK-AP-1 in human thyroid cells, suggesting a not apoptotic but radio-resistant role of PKC δ in human thyroid cells following IR.

Immunohistochemical localisation of protein tyrosine kinase receptors Tie-1 and Tie-2 in synovial tissue of rheumatoid arthritis: correlation with angiogenesis and synovial proliferation

OBJECTIVE To investigate the involvement of Tie-1 and Tie-2, receptor tyrosine kinases required for angiogenesis, in synovial proliferation and angiogenesis of rheumatoid arthritis (RA). METHODS Synovial tissues from 10 patients with RA and three control subjects were analysed by double immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS Expression of Tie-1 and Tie-2 was seen in all synovia, but predominantly in papillary projected portions. In synovial lining cells, Tie-2 was expressed mainly in the basal layer and frequently colocalised with vimentin and proliferating cell nuclear antigen (PCNA), whereas Tie-1 was also expressed in the superficial layer. In stromal cells, Tie-2 immunoreactivity was restricted to vimentin positive fibroblast—but not macrophage derived cells, whereas Tie-1 expression was not dependent on the phenotype. Tie receptors were also highly expressed in the endothelium and surrounding pericytes of capillaries scattered over the papillary proliferated synovium without notable difference in the expression of the two receptors. Furthermore, Tie positive vessels often overexpressed PCNA. In normal synovia, expression of Tie receptors was restricted to the capillary endothelium. RT-PCR confirmed the expression of Tie-1 and Tie-2 in RA synovial tissues and also in the cultured synoviocytes. CONCLUSION The results suggest the possible involvement of overexpressed Tie-1 and Tie-2 in synovial lining and stromal cells in the pathophysiology of RA synovitis, probably through distinct mechanisms. Furthermore, expression of Tie receptors in actively growing vasculature may reflect the direct involvement of these receptors in angiogenesis and subsequent vascularisation.

Expression of parathyroid hormone-related protein in rat articular cartilage

Expression and localization of parathyroid hormone-related protein (PTHrP) in rat articular cartilage during fetal and postnatal periods were investigated by immunohistochemistry and in situ hybridization. PHTrP displayed distinct distribution and intensity of staining at different ages. In fetal (18-day-old) and young (3-week-old) rats, articular chondrocytes expressed abundant PTHrP throughout the entire thickness of cartilage. In contrast, in 60-week-old rats, PTHrP was expressed in a few articular chondrocytes of superficial and middle layers. Regulation of PTHrP and PTH/PTHrP receptor mRNA was also studied in cultured rat articular chondrocytes. Northern blot analysis revealed that both transforming growth factor-β (TGF-β), an important stimulator for chondrocyte proliferation and differentiation, and 10% fetal bovine serum (FBS) stimulated the expression of PTHrP mRNA with down-regulation of its receptor mRNA. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulated the expression of receptor without changes of PTHrP mRNA level. These results suggest that the changes in abundance and localization of PTHrP and its receptor may be directly involved in the cell growth and differentiation of articular cartilage.

Effects of interleukin-1 beta on insulin-like growth factor-I autocrine/paracrine axis in cultured rat articular chondrocytes.

OBJECTIVE--To clarify the interaction of tissue destruction and repair of articular cartilage during inflammation, the effects of interleukin-1 beta (IL-1 beta) on the expression of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins were examined. METHODS--Articular chondrocytes from five week rats were cultured in serum free medium treated with IL-1 beta (1-100 U/ml) for 24 hours. The concentration of IGF-1 in the conditioned medium was measured by RIA, and IGFBP were analysed by immunoligand blotting method. IGF-I receptors were also examined by [125I]IGF-I binding study. RESULTS--IL-1 beta induced the secretion of IGF-I and IGF-binding protein in chondrocytes; this was not inhibited by indomethacin (5 micrograms/ml). IL-1 beta also increased the number of IGF-I receptors but had no effect on receptor affinity. IL-1 beta inhibited chondrocyte proliferation, while exogenous IGF-I and growth hormone stimulated chondrocyte cell growth. IL-1 beta did not change IGF-I mRNA levels. CONCLUSION--IL-1 beta up-regulated the IGF-I autocrine/paracrine axis in cultured articular chondrocytes. These observations provide insight into the critical role played by IL-1 beta in tissue destruction and repair, and into the direct interaction between cytokines and growth factors associated with inflammatory arthropathy.

The Kaposi's sarcoma-associated herpesvirus K-bZIP protein represses transforming growth factor β signaling through interaction with CREB-binding protein

Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is involved in the pathogenesis of KS, primary effusion lymphoma, and multicentric Castlemans disease. K-bZIP, the protein encoded by the open reading frame K8 of KSHV, is a member of the basic region-leucine zipper family of transcription factors. We studied the mechanisms that underlie KSHV-induced oncogenesis by investigating whether K-bZIP perturbs signaling through transforming growth factor β (TGF-β), which inhibits proliferation of a wide range of cell types. K-bZIP repressed TGF-β-induced, Smad-mediated transcriptional activity and antagonized the growth-inhibitory effects of TGF-β. Since both K-bZIP and Smad are known to interact with CREB-binding protein (CBP), the effect of CBP on inhibition of Smad-mediated transcriptional activation by K-bZIP was examined. K-bZIP mutants, which lacked the CBP-binding site, could not repress TGF-β-induced or Smad3-mediated transcriptional activity. Overexpression of CBP restored K-bZIP-induced inhibition of Smad3-mediated transcriptional activity. Competitive interaction studies showed that K-bZIP inhibited the interaction of Smad3 with CBP. These results suggest that K-bZIP, through its binding to CBP, disrupts TGF-β signaling by interfering with the recruitment of CBP into transcription initiation complexes on TGF-β-responsive elements. We propose a possibility that K-bZIP may contribute to oncogenesis through its ability to promote cell survival by repressing TGF-β signaling.

Repression of Smad‐dependent transforming growth factor‐β signaling by Epstein‐Barr virus latent membrane protein 1 through nuclear factor‐κB

EBV‐encoded LMP‐1 is absolutely required for EBV transformation of cells. Previous studies showed that LMP‐1 is responsible for mediating resistance to the anti‐proliferative effects of TGF‐β that characterizes EBV‐transformed cells. To clarify the mechanisms of resistance to TGF‐β by LMP‐1, we examined the effect of expression of LMP‐1 on the activity of TGF‐β‐responsive promoters. Interestingly, LMP‐1 inhibited TGF‐β‐responsive promoters activity despite lack of direct interaction of LMP‐1 and Smad proteins, intracellular signaling molecules in the TGF‐β signal transduction pathway. Although TGF‐β treatment increased the expression of p15, TGF‐β‐induced gene, this effect was counteracted by expression of LMP‐1. The repressive effect was mapped to the NF‐κB activation domains in the cytoplasmic carboxyl terminus of LMP‐1. Furthermore, LMP‐1‐mediated inhibition of TGF‐β‐responsive promoter was markedly restored after inhibition of NF‐κB activity. LMP‐1 failed to affect receptor‐dependent formation of heteromers containing Smad proteins as well as the DNA‐binding activity of Smad proteins. Overexpression of the transcriptional coactivator CBP and p300 abrogated the inhibitory effect of LMP‐1 on the TGF‐β‐responsive promoter. Our results suggest that LMP‐1 represses the TGF‐β signaling through the NF‐κB signaling pathway at transcriptional level by competing for a limited pool of transcriptional coactivators. These results enhance our understanding of the molecular mechanisms of viral pathogenesis in EBV‐associated malignancies.