Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice

In rheumatoid synovial tissues, synovial fibroblasts are activated by proinflammatory cytokines and proliferate to develop hyperplastic pannus tissues, which irreversibly damage the affected joints. We recently reported that the cyclin-dependent kinase inhibitors p16(INK4a) and p21(Cip1) are not expressed in vivo in rheumatoid synovial fibroblasts, but are readily inducible in vitro. This observation was followed by the successful treatment of rat adjuvant arthritis by local p16(INK4a) gene transfer, showing that the inhibition of the cell cycle of the synovial cells ameliorates the arthritis. In this study, we show that another animal model of rheumatoid arthritis, murine collagen-induced arthritis, can be effectively treated by local gene transfer of p21(Cip1) as well as that of p16(INK4a). The anti-arthritic effects were observed even when the treatment was conducted after the arthritis had developed. Furthermore, the effects included suppression of the expression of proinflammatory cytokines such as IL-1ss, IL-6, and TNF-alpha. Our results demonstrate that the ectopic expression of cyclin-dependent kinase inhibitors not only prevents synovial overgrowth but also ameliorates the proinflammatory milieu in the affected joints. The induction of p21(Cip1) in rheumatoid synovial tissues by pharmacological agents may also be an effective strategy to treat rheumatoid arthritis.     

Heterologous expression of the human cyclin-dependent kinase inhibitor p21Cip1 in the fission yeast, Schizosaccharomyces pombe reveals a role for PCNA in the chk1+ cell cycle checkpoint pathway.

Fission yeast cells expressing the human gene encoding the cyclin-dependent kinase inhibitor protein p21Cip1 were severely compromised for cell cycle progress. The degree of cell cycle inhibition was related to the level of p21Cip1 expression. Inhibited cells had a 2C DNA content and were judged by cytology and pulsed field gel electrophoresis to be in the G2 phase of the cell cycle. p21Cip1 accumulated in the nucleus and was associated with p34cdc2 and PCNA. Thus, p21Cip1 interacts with the same targets in fission yeast as in mammalian cells. Elimination of p34cdc2 binding by mutation within the cyclin-dependent kinase binding domain of p21Cip1 exaggerated the cell cycle delay phenotype. By contrast, elimination of PCNA binding by mutation within the PCNA-binding domain completely abolished the cell cycle inhibitory effects. Yeast cells expressing wild-type p21Cip1 and the mutant form that is unable to bind p34cdc2 showed enhanced sensitivity to UV. Cell cycle inhibition by p21Cip1 was largely abolished by deletion of the chk1+ gene that monitors radiation damage and was considerably enhanced in cells deleted for the rad3+ gene that monitors both DNA damage and the completion of DNA synthesis. Overexpression of PCNA also resulted in cell cycle arrest in G2 and this phenotype was also abolished by deletion of chk1+ and enhanced in cells deleted for rad3+. These results formally establish a link between PCNA and the products of the rad3+ and chk1+ checkpoint genes.     

Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis.

Synovial tissue affected by rheumatoid arthritis is characterized by proliferation, which leads to irreversible cartilage and bone destruction. Current and experimental treatments have been aimed mainly at correcting the underlying immune abnormalities, but these treatments often prove ineffective in preventing the invasive destruction. We studied the expression of cyclin-dependent kinase inhibitors in rheumatoid synovial cells as a means of suppressing synovial cell proliferation. Synovial cells derived from hypertrophic synovial tissue readily expressed p16INK4a when they were growth-inhibited. This was not seen in other fibroblasts, including those derived from normal and osteoarthritis-affected synovial tissues. In vivo adenoviral gene therapy with the p16INK4a gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis. Thus, the induction of p16INK4a may provide a new approach to the effective treatment of rheumatoid arthritis.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.     

Akt-Dependent Phosphorylation of p21Cip1 Regulates PCNA Binding and Proliferation of Endothelial Cells

The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21(Cip1) at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21(Cip1) at Thr 145 prevents the complex formation of p21(Cip1) with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21(Cip1) at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21(Cip1) and attenuates the Cdk2 inhibitory activity of p21(Cip1). Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21(Cip1) phosphorylation is not caused by altered intracellular localization of p21(Cip1). As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21(Cip1), whereas the nonphosphorylatable p21(Cip1) T145A construct arrests cells in G(0) phase. These data suggest that the modulation of p21(Cip1) cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.     

Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.     

Expression and regulation of inducible IkappaB kinase (IKK-i) in human fibroblast-like synoviocytes

IkappaB kinase (IKK) plays a key role in the regulation of nuclear factor kappaB (NF-kappaB). We previously demonstrated the expression of two kinases, IKK1 and IKK2, in fibroblast-like synoviocytes (FLS) and determined their functional consequences for inflammatory gene expression in vitro and in vivo. Recently, a novel inducible IkappaB kinase has been described, namely, IKK-i or IKK-epsilon, which is functionally and structurally distinct from constitutively expressed IKK1 and IKK2. Therefore, we investigated the expression and regulation of this novel kinase in FLS from patients with rheumatoid arthritis and osteoarthritis. Interestingly, constitutive gene expression and protein expression were observed in all cell lines examined. TNFalpha stimulation for 24 h increased IKK-i expression 7.2 +/- 1.8-fold in FLS (P < 0.02). IL-1 also significantly increased IKK-i gene expression. Time course experiments demonstrated that IKK-i gene expression increased within 3 h of TNFalpha stimulation and persisted for at least 24 h. Dose-response studies showed that as little as 1 ng/ml of TNFalpha increased IKK-i gene expression. Constitutive IKK-1 gene expression was also noted in rheumatoid arthritis, osteoarthritis, and normal synovium. This is the first report demonstrating constitutive expression and cytokine regulation of this novel kinase in primary human synovial cells.     

IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-alpha, IL-1beta, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.     

Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters

We examine the cell proliferation activity and expression of cyclin-dependent kinase inhibitors of the Cip/Kip family, p21Cip1, p27Kip1 and p57Kip2, in foetal hamster lungs to determine the expression patterns of the cyclin-dependent kinase inhibitors and to clarify the relationship between expression of the cyclin-dependent kinase inhibitors and lung development. Foetal hamster lungs on gestational days 12.5-16 (the day of birth) and adult lungs were fixed in 4% paraformaldehyde. Frozen sections were immunostained for the cyclin-dependent kinase inhibitors, and examined by immunostaining for Ki-67 and bromodeoxyuridine to determine the proliferation activity of the foetal lungs. During the foetal period, cell proliferation activity, as analysed by Ki-67 or bromodeoxyuridine labelling, decreased with development of the lung. In contrast to the gradual decrease of cell proliferation activity, cells with p27Kip1 immunoreactivity increased with development. On the other hand, p21Cip1-positive cells were most prominent around gestational day 14.5, while after birth positive cells decreased markedly. A few p57Kip2-positive cells were detected in the bronchiolar epithelium on gestational day 14.5. Western blotting analyses confirmed these immunostaining patterns. Thus, the levels of the cyclin-dependent kinase inhibitors of the Cip/Kip family are modulated in the lungs during the foetal period, and each shows a unique expression pattern. The cyclin-dependent kinase inhibitors may play roles not only in regulating cell proliferation activity but also in regulating other functions such as differentiation in the lung during the foetal period.     

Efficient down-regulation of cyclin A-associated activity and expression in suspended primary keratinocytes requires p21(Cip1)

When suspended in methylcellulose, primary mouse keratinocytes cease proliferation and differentiate. Suspension also reduces the activity of the cyclin-dependent kinase cdk2, an important cell cycle regulatory enzyme. To determine how suspension modulates these events, we examined its effects on wild-type keratinocytes and keratinocytes nullizygous for the cdk2 inhibitor p21(Cip1). After suspension of cycling cells, amounts of cyclin A (a cdk2 partner), cyclin A mRNA, and cyclin A-associated activity decreased much more rapidly in the presence than in the absence of p21(Cip1). Neither suspension nor p21(Cip1) status affected the stability of cyclin A mRNA. Loss of p21(Cip1) reduced the capacity of suspended cells to growth arrest, differentiate, and accumulate p27(Kip1) (a second cdk2 inhibitor) and affected the composition of E2F DNA binding complexes. Cyclin A-cdk2 complexes in suspended p21(+/+) cells contained p21(Cip1) or p27(Kip1), whereas most of the cyclin A-cdk2 complexes in p21(-/-) cells lacked p27(Kip1). Ectopic expression of p21(Cip1) allowed p21(-/-) keratinocytes to efficiently down-regulate cyclin A and differentiate when placed in suspension. These findings show that p21(Cip1) mediates the effects of suspension on numerous processes in primary keratinocytes including cdk2 activity, cyclin A expression, cell cycle progression, and differentiation.     

Negative regulation of ASK1 by p21Cip1 involves a small domain that includes Serine 98 that is phosphorylated by ASK1 in vivo

The cyclin-dependent kinase inhibitor p21(Cip1) regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21(Cip1). Here we show that small deletions of p21(Cip1) around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21(Cip1) and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21(Cip1) is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21(Cip1), predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21(Cip1) to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98.     

Production of monocyte chemoattractant protein-1 by inflamed synovial tissue and cultured synoviocytes.

This study analyzes the expression of monocyte chemoattractant protein-1 (MCP-1) by inflamed synovial tissue and defines its regulation in cultured synoviocytes. Synoviocytes from patients with rheumatoid arthritis and osteoarthritis express the 0.7-kb MCP-1 mRNA. Stimulation of synoviocytes with IL-1, TNF-alpha, LPS, platelet-derived growth factor, and transforming growth factor-beta-1, but not with basic fibroblast growth factor causes a marked increase in MCP-1 mRNA levels. Expression of the MCP-1 gene is inducible by activators of the protein kinase A (cAMP) and C (PMA) signal transduction pathways and is differentially regulated by the steroids dexamethasone and retinoic acid. Cultured synoviocytes de novo synthesize 12-, 15-, and 15.2-kDa MCP-1 proteins, which increase after stimulation with IL-1. Synovial tissues from donors without joint disease and from patients with rheumatoid or osteoarthritis were analyzed for MCP-1 mRNA expression by in situ hybridization. In these samples MCP-1 mRNA expressing cells were predominantly found in the sublining cell layers, whereas specimens of normal synovial tissue contained only few positive cells. These results identify synoviocytes as a source of MCP-1. Its expression is controlled by peptide regulatory factors that are known to be present in arthritic joints. Detection of cells producing MCP-1 mRNA in synovial tissues from patients with arthritis shows that this gene is expressed in vivo and suggests that MCP-1 can play a role in recruiting monocytes in joint inflammation.