Dysregulated FOXO transcription factors in articular cartilage in aging and osteoarthritis.

OBJECTIVE: Aging is a major risk factor for osteoarthritis (OA). Forkhead-box class O (FoxO) transcription factors regulate mechanisms of cellular aging, including protein quality control, autophagy and defenses against oxidative stress. The objective of this study was to analyze FoxO transcription factors in normal, aging and OA cartilage. DESIGN: Knee joints from humans ages 23-90 and from mice at the age of 4-24 months and following surgically induced OA were analyzed for expression of FoxO proteins. Regulation of FoxO protein expression and activation was analyzed in cultured chondrocytes. RESULTS: Human cartilage expressed FOXO1 and FOXO3 but not FOXO4 proteins. FOXO1 and FOXO3 were more strongly expressed the superficial and mid zone as compared to the deep zone and were mainly localized in nuclei. During human joint aging, expression of FOXO1 and FOXO3 was markedly reduced in the superficial zone of cartilage regions exposed to maximal weight bearing. In OA cartilage, chondrocyte clusters showed strong FOXO phosphorylation and cytoplasmic localization. Similar patterns of FOXO expression in normal joints and changes in aging and OA were observed in mouse models. In cultured chondrocytes, IL-1ß and TNF-α suppressed FOXO1, while TGF-ß and PDGF increased FOXO1 and FOXO3 expression. FOXO1 and FOXO3 phosphorylation was increased by IL-1ß, PDGF, bFGF, IGF-1, and the oxidant t-BHP. CONCLUSIONS: Normal articular cartilage has a tissue specific signature of FoxO expression and activation and this is profoundly altered in aging and OA in humans and mice. Changes in FoxO expression and activation may be involved in cartilage aging and OA.

Responses of microRNAs 124a and 223 following spinal cord injury in mice.

STUDY DESIGN: We investigated microRNA (miRNA) expression after spinal cord injury (SCI) in mice. OBJECTIVES: The recent discovery of miRNAs suggests a novel regulatory control over gene expression during plant and animal development. MiRNAs are short noncoding RNAs that suppress the translation of target genes by binding to their mRNAs, and play a central role in gene regulation in health and disease. The purpose of this study was to examine miRNA expression after SCI. SETTING: Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University. METHODS: We examined the expression of miRNA (miR)-223 and miR-124a in a mouse model at 6 h, 12 h, 1 day, 3 days and 7 days after SCI using quantitative PCR. The miRNA expression was confirmed by in situ hybridization. RESULTS: Quantitative PCR revealed two peaks of miR-223 expression at 6 and 12 h and 3 days after SCI. MiR-124a expression decreased significantly from 1 day to 7 days after SCI. In situ hybridization demonstrated the presence of miR-223 around the injured site. However, miR-124a, which was present in the normal spinal cord, was not observed at the injured site. CONCLUSION: Our results indicate a time-dependent expression pattern of miR-223 and miR-124a in a mouse model of SCI. In this study, the time course of miRNA-223 expression may be related to inflammatory responses after SCI, and the time course of decreased miR-124a expression may reflect cell death.

Inhibition of histone deacetylase down-regulates the expression of hypoxia-induced vascular endothelial growth factor by rheumatoid synovial fibroblasts.

OBJECTIVE: To investigate the effect of FK228 on the in vitro expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) by rheumatoid arthritis synovial fibroblasts (RASFs), and on the in vivo expression of VEGF and angiogenesis in the synovial tissue of mice with collagen-antibody-induced arthritis (CAIA). METHODS: RASFs were stimulated with IL-1beta and TNFalpha and then incubated under hypoxia (1 % O(2)) with various concentrations of FK228. The effects of FK228 on the expression of HIF-1alpha and VEGF mRNA were examined by quantitative real-time PCR. Changes in HIF-1alpha protein expression and the secretion of VEGF protein into the culture medium were examined by Western blot analysis and ELISA, respectively. Immunohistochemical analysis was carried out to investigate the expression and distribution of VEGF in synovial tissues of CAIA mice. RESULTS: The cytokine-stimulated expression of HIF-1alpha and VEGF mRNA was inhibited by FK228 in a dose-dependent manner. FK228 also reduced the expression of HIF-1alpha and VEGF protein. Intravenous administration of FK228 (2.5 mg/kg) suppressed VEGF expression, and also blocked angiogenesis in the synovial tissue of CAIA. CONCLUSION: FK228 may exhibit a therapeutic effect on RA by inhibition of angiogenesis through down-regulation of angiogenesis related factors, HIF-1alpha and VEGF.

Acid-catalyzed rearrangement of aryl-substituted homobenzoquinone epoxides.

The BF3-catalyzed reactions of diphenyl-substituted and endo-monophenyl-substituted homobenzoquinone epoxides proceeded through a regioselective oxirane ring opening followed by participation of a pi-aryl transannular cyclization to give the tricyclic diketo alcohols. The conformationally semirigid ethano-bridged diphenyl-substituted homologues also provided similar diketo alcohols and the subsequent ring-expanded cycloheptenedione (via a subsequent 1,2-acyl migration associated with cyclopropane ring opening), depending on the methyl-substitution pattern of the quinone frame. However, the exo-monophenyl-substituted and the rigid biphenyl-2,2'-diyl-substituted homobenzoquinone epoxides essentially remained unchanged.

Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB.

The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.

A transcriptional switch mediated by cofactor methylation.

We describe a molecular switch based on the controlled methylation of nucleosome and the transcriptional cofactors, the CREB-binding proteins (CBP)/p300. The CBP/p300 methylation site is localized to an arginine residue that is essential for stabilizing the structure of the KIX domain, which mediates CREB recruitment. Methylation of KIX by coactivator-associated arginine methyltransferase 1 (CARM1) blocks CREB activation by disabling the interaction between KIX and the kinase inducible domain (KID) of CREB. Thus, CARM1 functions as a corepressor in cyclic adenosine monophosphate signaling pathway via its methyltransferase activity while acting as a coactivator for nuclear hormones. These results provide strong in vivo and in vitro evidence that histone methylation plays a key role in hormone-induced gene activation and define cofactor methylation as a new regulatory mechanism in hormone signaling.

Stimulation of DNA polymerase alpha activity by cdk2-phosphorylated Rb protein.

We propose a new role of retinoblastoma protein as a cell growth activator in its phosphorylated form. The hyper-phosphorylated retinoblastoma protein generated by the action of cdk2/cyclin E strongly stimulated the activity of DNA polymerase alpha, but did not stimulate DNA polymerases delta, epsilon, or primase. But, cdk4/cyclin D-phosphorylated retinoblastoma protein showed little stimulation. Hyper-phosphorylated retinoblastoma protein interacted with the catalytic subunit of DNA polymerase alpha, and stabilised DNA polymerase alpha from heat inactivation at 45 degrees C. These results suggest that in G1 phase, hypo-phosphorylated retinoblastoma protein suppresses the progression of cell cycle as a transcription inhibitor, but that after phosphorylation by cdk2/cyclin E at the G1/S boundary, hyper-phosphorylated retinoblastoma protein acts as a cell-cycle promoter by optimising the DNA polymerase alpha reaction.

Suppression of transthyretin expression by ribozymes: a possible therapy for familial amyloidotic polyneuropathy.

Familial amyloidotic polyneuropathy type 1 (FAP) is an autosomal-dominantly inherited disorder with systemic deposition of a variant transthyretin (TTR). We attempted to suppress TTR production by ribozyme degradation of TTR mRNA. Hammerhead and hairpin ribozymes cleaved TTR mRNA at specific individual sites in vitro. A ribozyme targeting a variant TTR (E61K) degraded the variant mRNA, but not a wild-type mRNA. These ribozymes also reduced the amounts of TTR mRNA and protein in HepG2 cells and COS-1 cells transfected with TTR-E61K cDNA. Ribozymes might be studied further as a potential treatment for FAP.

The CREB family of activators is required for endochondral bone development.

We have evaluated the importance of the CREB family of transcriptional activators for endochondral bone formation by expressing a potent dominant negative CREB inhibitor (A-CREB) in growth plate chondrocytes of transgenic mice. A-CREB transgenic mice exhibited short-limbed dwarfism and died minutes after birth, apparently due to respiratory failure from a diminished rib cage circumference. Consistent with the robust Ser133 phosphorylation and, hence, activation of CREB in chondrocytes within the proliferative zone of wild-type cartilage during development, chondrocytes in A-CREB mutant cartilage exhibited a profound decrease in proliferative index and a delay in hypertrophy. Correspondingly, the expression of certain signaling molecules in cartilage, most notably the Indian hedgehog (Ihh) receptor patched (Ptch), was lower in A-CREB expressing versus wild-type chondrocytes. CREB appears to promote Ptch expression in proliferating chondrocytes via an Ihh-independent pathway; phospho-CREB levels were comparable in cartilage from Ihh(-/-) and wild-type mice. These results demonstrate the presence of a distinct signaling pathway in developing bone that potentiates Ihh signaling and regulates chondrocyte proliferation, at least in part, via the CREB family of activators.

Recognition of YKL-39, a human cartilage related protein, as a target antigen in patients with rheumatoid arthritis.

OBJECTIVE: To investigate whether autoimmunity to YKL-39, a recently cloned cartilage protein, occurs in patients with rheumatoid arthritis (RA). METHODS: Autoantibody to YKL-39 was assayed by enzyme linked immunosorbent assay (ELISA) and western blotting in serum samples from patients with RA, systemic lupus erythematosus (SLE), and healthy donors, using recombinant YKL-39 protein. This reactivity was compared with that against a YKL-39 homologue, YKL-40 (human cartilage gp-39/chondrex), which has been reported to be an autoantigen in RA. RESULTS: Autoantibody to YKL-39 was detected in seven of 87 patients with RA (8%), but not in serum samples from patients with SLE or healthy donors. YKL-40 reactivity was found in only one of 87 RA serum samples (1%), with no cross reactivity to YKL-39. CONCLUSION: The existence of anti-YKL-39 antibody in a subset of patients with RA is reported here for the first time. Further, it was shown that the immune response to YKL-39 was independent of that to YKL-40. Clarification of the antibody and T cell responses to autoantigens derived from chondrocyte, cartilage, or other joint components may lead to a better understanding of the pathophysiology of joint destruction in patients with RA.

Lovastatin does not correct the accumulation of very long-chain fatty acids in tissues of adrenoleukodystrophy protein-deficient mice.

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A reductase, normalizes the very long-chain fatty acids (VLCFA) concentrations in fibroblasts and plasma from patients with X-linked adrenoleukodystrophy (X-ALD). The effects of lovastatin on the accumulation of VLCFA in tissues of adrenoleukodystrophy protein (ALDP)-deficient mice were assessed. ALDP-deficient mice were fed chow with 0.01-0.1% lovastatin for 4-8 weeks. The VLCFA concentrations in the plasma, brain, spinal cord, liver and kidneys were measured. Treatment with 0.1% lovastatin significantly reduced body weight and total cholesterol in the plasma of ALDP-deficient mice. Treatment with lovastatin, however, did not correct the accumulation of VLCFA in the plasma or tissues, including the brain and spinal cord. Lovastatin does not affect the accumulation of VLCFA in ALDP-deficient tissues in mice.

Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor.

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol alpha) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol alpha. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as delta and epsilon; it also showed no effect even on the pol alpha activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 microm. Dehydroaltenusin-induced inhibition of mammalian pol alpha activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol alpha, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol alpha. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol alpha, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.

Expression of neurotrophins and their receptors (TRK) during fracture healing.

To clarify the roles of neurotrophins and their receptors in bone formation, expression of neurotrophins and their receptors (TRK) in a model of mouse fracture healing was investigated. A total of 120 male ICR mice were studied. The right eighth rib of 70 mice was fractured. For sham operation as a control, the right eighth rib of 50 mice was similarly exposed but not fractured. Localization of TRKA, TRKB, and TRKC in a rectangular region of the rib together with surrounding soft tissues was investigated by immunostaining. Localizations of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) at the fracture callus were also investigated by immunostaining, and their mitochondrial RNA (mRNA) expressions were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). As a result, we observed two types of neurotrophin receptors in the bone forming area: immunostaining by anti-TRKA was observed in almost all bone forming cells, and staining with anti-TRKC was observed in osteoblast-like cells and hypertrophic chondrocytes, but no staining was observed with anti-TRKB. On the other hand, localization of NGF was observed in almost all bone forming cells, localization of BDNF was observed in osteoblast-like cells, and localization of NT-3 was observed in osteoblast-like cells and hypertrophic chondrocytes at the fracture callus. Expression levels of the mRNA of three neurotrophins in the fractured rib were increased during the process of healing, especially those of NGF and NT-3, which peaked at 2 days after the fracture. The level of BDNF mRNA increased gradually over 8 days. These findings show that neurotrophins and their receptors were expressed in bone forming cells, and suggest that they are involved in the regulation of bone formation as an autocrine and paracrine factor in vivo.

Increased osteocyte apoptosis during the development of femoral head osteonecrosis in spontaneously hypertensive rats.

We investigated the presence of osteocyte apoptosis in the necrotic trabeculae of the femoral head of spontaneously hypertensive rat (SHR) using the in situ nick end labeling (TUNEL) method and transmission electron microscopy. The occurrence of osteonecrosis and ossification disturbance was significantly higher in SHR compared with Wistar Kyoto (WKY) rats, and Wistar (WT) rats used as control animals (P < 0.01). A high population of TUNEL positive osteocytes was detected mainly in 10- and 15-week-old SHRs. Sectioned examination of the femoral head of SHRs and WKY rats by electron microscopy revealed apoptotic cell appearances such as aggregation of chromatin particles and lipid formation. In contrast, a positive reaction was significantly lower in osteocytes in the femoral heads of WT rats (P < 0.01). Our results indicate that apoptosis forms an important component of the global pathologic process affecting the femoral head of SHR, which leads to osteonecrosis in this region.

Characterization of a CREB gain-of-function mutant with constitutive transcriptional activity in vivo.

The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator paralogs CREB-binding protein (CBP) and p300. CBP and p300, in turn, appear to mediate target gene induction via their association with RNA polymerase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide variety of stimuli, including hypoxia, UV irradiation, and growth factor addition, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of promoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-CBP complex formation or in the subsequent recruitment of the transcriptional apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wild-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene expression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the K(m) for PKA phosphorylation and thereby induces high levels of constitutive Ser133 phosphorylation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB strongly promoted differentiation of PC12 cells in concert with suboptimal doses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter.

The phosphorylation status of a cyclic AMP-responsive activator is modulated via a chromatin-dependent mechanism.

Cyclic AMP (cAMP) stimulates the expression of numerous genes via the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133. Ser133 phosphorylation, in turn, promotes recruitment of the coactivator CREB binding protein and its paralog p300, histone acetyltransferases (HATs) that have been proposed to mediate target gene activation, in part, by destabilizing promoter bound nucleosomes and thereby allowing assembly of the transcriptional apparatus. Here we show that although histone deacetylase (HDAC) inhibitors potentiate target gene activation via cAMP, they do not stimulate transcription over the early burst phase, during which CREB phosphorylation and CBP/p300 recruitment are maximal. Rather, HDAC inhibitors augment CREB activity during the late attenuation phase by prolonging CREB phosphorylation on chromosomal but, remarkably, not on extrachromosomal templates. In reconstitution studies, assembly of periodic nucleosomal arrays on a cAMP-responsive promoter template potently inhibited CREB phosphorylation by PKA, and acetylation of these template-bound nucleosomes by p300 partially rescued CREB phosphorylation by PKA. Our results suggest a novel regulatory mechanism by which cellular HATs and HDACs modulate the phosphorylation status of nuclear activators in response to cellular signals.

Glutamate enhances phosphorylation of neurofilaments in cerebellar granule cell culture.

In amyotrophic lateral sclerosis (ALS), an abnormal increase of glutamate in the central nervous system indicates that it may play a key role in motor neuron death. The neuronal accumulation of phosphorylated neurofilaments (NFs) suggests an alteration of phosphorylation of NFs is also involved. Rat cerebellar granule cells (CGCs) are sensitive to glutamate neurotoxicity and provide a suitable model system for clarifying its mechanisms. Using cultured CGCs, we investigated the relationship between glutamate neurotoxicity and the phosphorylation of NFs. Because glutamate showed a dose-dependent neurotoxicity for CGCs, we adopted a 10 microM glutamate treatment, which produced no acute neurotoxicity during the experiments. The number of phosphorylated heavy subunits of neurofilaments (NF-Hs) increased to approximately twice that of the control after 72 h, although the total number of NF-Hs remained constant throughout the experiment. The phosphorylation of NF-Hs was significantly suppressed by the AMPA-receptor antagonist CNQX, but not by the NMDA-receptor antagonist MK-801. Our findings therefore suggest that exposure to a low concentration of glutamate enhances the phosphorylation of NF-Hs, mainly via the AMPA receptor.

Increased levels of nitrite/nitrate in the cerebrospinal fluid of patients with subarachnoid hemorrhage.

The role of nitric oxide (NO) in the mechanism of delayed cerebral vasospasm (VS) after subarachnoid hemorrhage (SAH) was investigated by analyzing the stable metabolites of NO, nitrite and nitrate, by the Griess method in the cerebrospinal fluid (CSF) and venous blood of 29 patients with SAH, the CSF of 22 control patients, and venous blood from eight normal subjects. VS was defined as diffuse and severe angiographical vasospasm detected by angiography performed around days 7-9 after the onset. Six of the 29 patients had VS. The nitrite/nitrate levels in the blood of patients with SAH were almost within the range of those in normal subjects, but the levels in the CSF of patients with SAH were significantly higher than those of the control group. Patients with VS after SAH had significantly lower levels in the CSF than patients without VS on days 7-9, when VS is most likely to occur. These observations suggest that NO production in the CSF environment occurs following SAH, but possibly may not provoke VS.

Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner.

Homeobox (hox) proteins have been shown to regulate cell fate and segment identity by promoting the expression of specific genetic programs. In contrast to their restricted biological action in vivo, however, most homeodomain factors exhibit promiscuous DNA binding properties in vitro, suggesting a requirement for additional cofactors that enhance target site selectivity. In this regard, the pbx family of homeobox genes has been found to heterodimerize with and thereby augment the DNA binding activity of certain hox proteins on a subset of potential target sites. Here we examine the transcriptional properties of a forced hox-pbx heterodimer containing the pancreas-specific orphan homeobox factor pdx fused to pbx-1a. Compared to the pdx monomer, the forced pdx-pbx1a dimer, displayed 10- to 20-fold-higher affinity for a consensus hox-pbx binding site but was completely unable to bind a hox monomer recognition site. The pdx-pbx dimer stimulated target gene expression via an N-terminal trans-activation domain in pdx that interacts with the coactivator CREB binding protein. The pdx-pbx dimer was also found to repress transcription via a C-terminal domain in pbx-1a that associates with the corepressors SMRT and NCoR. The transcriptional properties of the pdx-pbx1 complex appear to be regulated at the level of alternative splicing; a pdx-pbx polypeptide containing the pbx1b isoform, which lacks the C-terminal extension in pbx1a, was unable to repress target gene expression via NCoR-SMRT. Since pbx1a and pbx1b are differentially expressed in endocrine versus exocrine compartments of the adult pancreas, our results illustrate a novel mechanism by which pbx proteins may modulate the expression of specific genetic programs, either positively or negatively, during development.