Sex-Specific Skeletal Muscle Gene Expression Responses to Exercise Reveal Novel Direct Mediators of Insulin Sensitivity Change.

BACKGROUND: Understanding the causal pathways, systems, and mechanisms through which exercise impacts human health is complex. This study explores molecular signaling related to whole-body insulin sensitivity (Si) by examining changes in skeletal muscle gene expression. The analysis considers differences by biological sex, exercise amount, and exercise intensity to identify potential molecular targets for developing pharmacologic agents that replicate the health benefits of exercise. METHODS: The study involved 53 participants from the STRRIDE I and II trials who completed eight months of aerobic training. Skeletal muscle gene expression was measured using Affymetrix and Illumina technologies, while pre- and post-training Si was assessed via an intravenous glucose tolerance test. A novel gene discovery protocol, integrating three literature-derived and data-driven modeling strategies, was employed to identify causal pathways and direct causal factors based on differentially expressed transcripts associated with exercise intensity and amount. RESULTS: In women, the transcription factor targets identified were primarily influenced by exercise amount and were generally inhibitory. In contrast, in men, these targets were driven by exercise intensity and were generally activating. Transcription factors such as ATF1, CEBPA, BACH2, and STAT1 were commonly activating in both sexes. Specific transcriptional targets related to exercise-induced Si improvements included TACR3 and TMC7 for intensity-driven effects, and GRIN3B and EIF3B for amount-driven effects. Two key signaling pathways mediating aerobic exercise-induced Si improvements were identified: one centered on estrogen signaling and the other on phorbol ester (PKC) signaling, both converging on the epidermal growth factor receptor (EGFR) and other relevant targets. CONCLUSIONS: The signaling pathways mediating Si improvements from aerobic exercise differed by sex and were further distinguished by exercise intensity and amount. Transcriptional adaptations in skeletal muscle related to Si improvements appear to be causally linked to estrogen and PKC signaling, with EGFR and other identified targets emerging as potential skeletal muscle-specific drug targets to mimic the beneficial effects of exercise on Si.

Alpha defensin-1 attenuates surgically induced osteoarthritis in association with promoting M1 to M2 macrophage polarization.

OBJECTIVE: Macrophages play an important part in the pathogenesis of osteoarthritis (OA). Our objective was to determine the effects of α-defensin-1 on macrophage polarization and consequently OA. METHODS: OA synovial tissue and synovial fluid were assessed for the presence of M1 (CD68+CD16+CD206-) and M2 (CD68+CD206+CD16-) macrophages by flow cytometry. M0, M1, and M2 macrophages were co-cultured with OA chondrocytes to determine their influence on chondrogenic phenotype. Polarization of THP-1 activated monocytes from M1 to M2 in response to α-defensin-1 was evaluated by flow cytometry, RT-PCR and RNA sequencing. Effects of intra-articular α-defensin-1 in vivo were evaluated in a rat meniscal/ligamentous injury (MLI) model. RESULTS: The quantity of M1 exceeded M2 polarized macrophages in human OA synovial tissue (mean difference 26.1% [13.6-38.6%], P < 0.001) and fluid (mean difference 10.5% [5.0-16.1%], P = 0.003). M1 to M2 polarization in vitro was most effectively promoted with 10 ng/mL α-defensin-1. Compared with untreated macrophages, the α-defensin-1 polarized macrophages modified co-cultured OA chondrocytes from a pro-catabolic state to a pro-anabolic (regenerative-like) state based on expression of COL2A1, ACN, MMP3, MMP13 and ADAMTS5. Intra-articular α-defensin-1 decreased severity of cartilage damage and synovitis in the MLI rat model. RNAseq analyses suggested insulin and Toll-like receptor signaling pathways in the chondroprotective α-defensin-1 mechanism of action. CONCLUSION: α-defensin-1 promotes M1 to M2 macrophage polarization in vitro, has beneficial effects on chondrocytes indirectly via M2 macrophage polarization, and attenuates the severity of OA in vivo, suggesting it might be a candidate treatment for OA.

Anti-inflammatory effects of naproxen sodium on human osteoarthritis synovial fluid immune cells.

OBJECTIVE: To evaluate the anti-inflammatory effects of clinically relevant naproxen sodium (Nx) concentrations on human monocyte-derived macrophages in a controlled in vitro system and human primary synovial fluid (SF) cells. DESIGN: Using phorbol 12-myristate 13-acetate, THP-1 human monocytic cells were differentiated into mature monocyte-derived macrophages in vitro then treated with Nx pre- or post-activating an inflammatory response with lipopolysaccharide (LPS) and hyaluronan (HA) fragments (n = 8/group). Cell culture supernatants were assessed for NF-κB activity and prostaglandin E2 (PGE2), indicating cyclooxygenase enzyme activity. Under Duke IRB approval, primary human SF cells were collected at the time of knee joint replacement (n = 19 individuals) for osteoarthritis (OA), and cultured with LPS, HA and Nx; SF cells were characterized by polychromatic flow cytometry for cell surface markers and intracellular cytokines. RESULT: Compared to placebo treatment of THP-1 cells, low dose Nx (corresponding 27.5-440 mg/L orally) added both pre- and post-activation with LPS/HA, significantly reduced NF-κB activity and PGE2: mean reduction to 73%, 61%, 17% and 10% of placebo, respectively. LPS/HA treatment of primary OA SF cells significantly increased the number of IL-1ß producing primary monocytes and macrophages, and by 24 h the overall production of secreted cytokines (IL-1ß, IL-6, IL8, and TNF-α). Low dose Nx reduced the percentage of IL-1ß producing primary monocytes and macrophages. CONCLUSION: LPS/HA induced inflammation of THP-1 monocytic and primary human SF cells. Low dose Nx both prevented and reduced inflammatory responses of a human monocytic cell line and reduced IL-1ß production by primary human SF monocytes and macrophages.

Synovial and systemic pharmacokinetics (PK) of triamcinolone acetonide (TA) following intra-articular (IA) injection of an extended-release microsphere-based formulation (FX006) or standard crystalline suspension in patients with knee osteoarthritis (OA).

OBJECTIVE: Intra-articular (IA) corticosteroids relieve osteoarthritis (OA) pain, but rapid absorption into systemic circulation may limit efficacy and produce untoward effects. We compared the pharmacokinetics (PK) of IA triamcinolone acetonide (TA) delivered as an extended-release, microsphere-based formulation (FX006) vs a crystalline suspension (TAcs) in knee OA patients. METHOD: This Phase 2 open-label study sequentially enrolled 81 patients who received a single IA injection of FX006 (5 mL, 32 mg delivered dose, N = 63) or TAcs (1 mL, 40 mg, N = 18). Synovial fluid (SF) aspiration was attempted in each patient at baseline and one post-IA-injection visit (FX006: Week 1, Week 6, Week 12, Week 16 or Week 20; TAcs: Week 6). Blood was collected at baseline and multiple post-injection times. TA concentrations (validated LC-MS/MS, geometric means (GMs)), PK (non-compartmental analysis models), and adverse events (AEs) were assessed. RESULTS: SF TA concentrations following FX006 were quantifiable through Week 12 (pg/mL: 231,328.9 at Week 1; 3590.0 at Week 6; 290.6 at Week 12); post-TAcs, only two of eight patients had quantifiable SF TA at Week 6 (7.7 pg/mL). Following FX006, plasma TA gradually increased to peak (836.4 pg/mL) over 24 h and slowly declined to <110 pg/mL over Weeks 12-20; following TAcs, plasma TA peaked at 4 h (9628.8 pg/mL), decreased to 4991.1 pg/mL at 24 h, and was 149.4 pg/mL at Week 6, the last post-treatment time point assessed. AEs were similar between groups. CONCLUSION: In knee OA patients, microsphere-based TA delivery via a single IA injection prolonged SF joint residency, diminished peak plasma levels, and thus reduced systemic TA exposure relative to TAcs.

Direct in vivo evidence of activated macrophages in human osteoarthritis.

OBJECTIVE: Through binding to folate receptor-ß (FR-ß), the new (99m)Tc-EC20 (Etarfolatide) imaging technique detects activated but not resting macrophages in vivo. The goal of this study was to investigate macrophage-related inflammation in osteoarthritis (OA). METHODS: Twenty-five individuals (50 knees) with symptomatic OA of at least one knee underwent SPECT-CT imaging of both knees and planar imaging of the whole body after injection of Etarfolatide. Scans and knee radiographs were scored blinded to clinical information including knee and other joint site pain severity. Measures of association controlled for age, gender, body mass index (BMI) and employed repeated measures to adjust for correlation between knees. DESIGN: Activated macrophages were present in the majority (76%) of knees. The quantity of knee-related macrophages was significantly associated with knee pain severity (R = 0.60, P < 0.0001) and radiographic knee OA severity including joint space narrowing (R = 0.68, P = 0.007), and osteophyte (R = 0.66, P = 0.001). Macrophages were also localized to joints commonly affected by OA including hand finger joints (12%), thumb bases (28%), shoulders (26%), great toes (18%) and ankles (12%). The presence of joint pain at fingers, wrists, ankles and great toes was significantly positively associated with presence of activated macrophages at these sites (P < 0.0001-0.04). CONCLUSIONS: This study provides the first direct in vivo evidence for macrophage involvement in OA in a substantial proportion of human knees. The association of quantity of activated macrophages with radiographic knee OA severity and joint symptoms suggests that drugs targeting macrophages and macrophage-associated inflammatory pathways may have the potential to be both symptom and structure modifying.

In vivo luminescent imaging of NF-κB activity and NF-κB-related serum cytokine levels predict pain sensitivities in a rodent model of peripheral neuropathy.

BACKGROUND: Methods for the detection of the temporal and spatial generation of painful symptoms are needed to improve the diagnosis and treatment of painful neuropathies and to aid preclinical screening of molecular therapeutics. METHODS: In this study, we utilized in vivo luminescent imaging of NF-κB activity and serum cytokine measures to investigate relationships between the NF-κB regulatory network and the presentation of painful symptoms in a model of neuropathy. RESULTS: The chronic constriction injury model led to temporal increases in NF-κB activity that were strongly and non-linearly correlated with the presentation of pain sensitivities (i.e. mechanical allodynia and thermal hyperalgesia). The delivery of NEMO-binding domain peptide reduced pain sensitivities through the inhibition of NF-κB activity in a manner consistent with the demonstrated non-linear relationship. Importantly, the combination of non-invasive measures of NF-κB activity and NF-κB-regulated serum cytokines produced a highly predictive model of both mechanical (R(2) = 0.86) and thermal (R(2) = 0.76) pain centred on the NF-κB regulatory network (NF-κB, IL-6, CXCL1). CONCLUSIONS: Using in vivo luminescent imaging of NF-κB activity and serum cytokine measures, this work establishes NF-κB and NF-κB-regulated cytokines as novel multivariate biomarkers of pain-related sensitivity in this model of neuropathy that may be useful for the rapid screening of novel molecular therapeutics.

Atlas of radiographic features of osteoarthritis of the ankle and hindfoot.

OBJECTIVE: To develop a radiographic atlas of osteoarthritis (OA) for use as a template and guide for standardized scoring of radiographic features of OA of the ankle and hindfoot joints. METHOD: Under Institutional Review Board approval, ankle and hindfoot images were selected from a cohort study and from among cases that underwent ankle radiography during a 6-month period at Duke University Medical Center. Missing OA pathology was obtained through supplementation of cases with the assistance of a foot and ankle specialist in Orthopaedic surgery and a musculoskeletal radiologist. Images were obtained and reviewed without patient identifying information. Images went through multiple rounds of review and final images were selected by consensus of the study team. For intra-rater and inter-rater reliability, the kappa statistic was calculated for two readings by three musculoskeletal radiologists, a minimum of two weeks apart, of ankle and hindfoot radiographs from 30 anonymized subjects. RESULTS: The atlas demonstrates individual radiographic features (osteophyte and joint space narrowing (JSN)) and Kellgren-Lawrence grade for all aspects of the talocrural (ankle joint proper) and talocalcaneal (subtalar) joints. Reliability of scoring based on the atlas was quite good to excellent for most features indicated. Additional examples of ankle joint findings are illustrated including sclerosis, os trigonum, subchondral cysts and talar tilt. CONCLUSIONS: It is anticipated that this atlas will assist with standardization of scoring of ankle and hindfoot OA by basic and clinical OA researchers.

Effectiveness of low-level laser therapy in patients with knee osteoarthritis: a systematic review and meta-analysis.

OBJECTIVE: To investigate the efficacy of low-level laser therapy (LLLT) treatment of knee osteoarthritis (KOA) by a systematic literature search with meta-analyses on selected studies. DESIGN: MEDLINE, EMBASE, ISI Web of Science and Cochrane Library were systematically searched from January 2000 to November 2014. Included studies were randomized controlled trials (RCTs) written in English that compared LLLT (at least eight treatment sessions) with sham laser in KOA patients. The efficacy effective size was estimated by the standardized mean difference (SMD). Standard fixed or random-effects meta-analysis was used, and inconsistency was evaluated by the I-squared index (I(2)). RESULTS: Of 612 studies, nine RCTs (seven double-blind, two single-blind, totaling 518 patients) met the criteria for inclusion. Based on seven studies, the SMD in visual analog scale (VAS) pain score right after therapy (RAT) (within 2 weeks after the therapy) was not significantly different between LLLT and control (SMD = -0.28 [95% CI = -0.66, 0.10], I(2) = 66%). No significant difference was identified in studies conforming to the World Association of Laser Therapy (WALT) recommendations (four studies) or on the basis of OA severity. There was no significant difference in the delayed response (12 weeks after end of therapy) between LLLT and control in VAS pain (five studies). Similarly, there was no evidence of LLLT effectiveness based on Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain, stiffness or function outcomes (five and three studies had outcome data right after and 12 weeks after therapy respectively). CONCLUSION: Our findings indicate that the best available current evidence does not support the effectiveness of LLLT as a therapy for patients with KOA.

Sustained intra-articular delivery of IL-1RA from a thermally-responsive elastin-like polypeptide as a therapy for post-traumatic arthritis.

Post-traumatic arthritis (PTA) is a rapidly progressive form of arthritis that develops due to joint injury, including articular fracture. Current treatments are limited to surgical restoration and stabilization of the joint; however, evidence suggests that PTA progression is mediated by the upregulation of pro-inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α). Although these cytokines provide potential therapeutic targets for PTA, intra-articular injections of anti-cytokine therapies have proven difficult due to rapid clearance from the joint space. In this study, we examined the ability of a cross-linked elastin-like polypeptide (xELP) drug depot to provide sustained intra-articular delivery of IL-1 and TNF-α inhibitors as a beneficial therapy. Mice sustained a closed intra-articular tibial plateau fracture; treatment groups received a single intra-articular injection of drug encapsulated in xELP. Arthritic changes were assessed 4 and 8 weeks after fracture. Inhibition of IL-1 significantly reduced the severity of cartilage degeneration and synovitis. Inhibition of TNF-α alone or with IL-1 led to deleterious effects in bone morphology, articular cartilage degeneration, and synovitis. These findings suggest that IL-1 plays a critical role in the pathogenesis of PTA following articular fracture, and sustained intra-articular cytokine inhibition may provide a therapeutic approach for reducing or preventing joint degeneration following trauma.

High prevalence of contralateral ankle abnormalities in association with knee osteoarthritis and malalignment.

OBJECTIVE: To evaluate ankle joint abnormalities in a knee osteoarthritis (OA) cohort. METHODS: Participants (n = 159) with symptomatic and radiographic OA in at least one knee underwent technetium-99m methylene diphosphonate bone scan (scored 0-3) of the ankles and forefeet. Knee radiographs were graded for OA features of joint space narrowing (JSN) and osteophyte (OST). Ankle symptoms and history of ankle injury were assessed by self-report. Knee alignment was measured from a long-limb radiograph. Ankle radiographs were obtained on those who returned for follow-up (n = 138) and were graded for ankle tibiotalar JSN and OST. DESIGN: Ankle scintigraphic abnormalities were frequent (31% of individuals, one-third bilateral). Ankle symptoms were reported by 23% of individuals and history of ankle injury by 24%. Controlling for gender, age, body mass index (BMI), and contralateral predictor, ankle scintigraphic abnormalities were associated with: ipsilateral ankle symptoms (P = 0.005); contralateral knee JSN (P = 0.001), knee OST (P = 0.006) and knee malalignment (P = 0.08); and history of ankle injury or surgery of either ankle (P < 0.0001). At follow-up, scintigraphic abnormalities of the ankle were strongly associated with presence of tibiotalar radiographic OA (P < 0.0001). CONCLUSIONS: Although considered rare, we observed a high prevalence of radiographic features of ankle OA in this knee OA cohort. History of overt ankle injury did not appear to account for the majority of ankle abnormalities. These results are consistent with a probable kinematic association of knee OA pathology and contralateral ankle abnormalities and suggest that interventions targeting mechanical factors may be needed to prevent ankle OA in the setting of knee OA.

Effects of intraarticular IL1-Ra for acute anterior cruciate ligament knee injury: a randomized controlled pilot trial (NCT00332254).

OBJECTIVE: To evaluate the clinical effectiveness of intraarticular IL-1 receptor antagonist (IL-1Ra) for anterior cruciate ligament (ACL) tear. METHODS: Eleven patients with acute ACL tear confirmed by magnetic resonance imaging (MRI) were randomized to receive a single intraarticular injection of IL-1Ra (anakinra 150 mg, n = 6) or equal volume of saline placebo (1 ml, n = 5). The double-blinded treatment was administered a mean 2 weeks after injury. Synovial fluid (SF) (n = 9 patients) and sera (all patients) were available at baseline (prior to injection) and immediately prior to surgery (mean 35 days later) and analyzed for SF IL-1α, IL-1ß, IL-1Ra and serum hyaluronan (HA), an indicator of synovial inflammation. The primary outcome, standardized Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire, was obtained at 0 (baseline), 4, and 14 days after injection. RESULTS: Compared with placebo, the IL-1Ra group had substantially greater improvement in key outcomes over 14 days (KOOS pain P = 0.001; activities of daily living P = 0.0015; KOOS sports function P = 0.0026; KOOS quality of life (QOL) P = 0.0048; and total KOOS P < 0.0001). There were no adverse reactions in either group. SF IL-1α (P = 0.05) and serum HA (P = 0.03), but not IL-1ß, or IL-1Ra, decreased significantly in the IL-1Ra but not the placebo treated patients. Compared with placebo, IL-1α was borderline significantly different in the IL-1Ra treated group (P = 0.06). CONCLUSIONS: Administered within the first month following severe knee injury, IL-1Ra reduced knee pain and improved function over a 2-week interval. This promising proof of concept study provides a new paradigm for studies of acute joint injury and suggests that a larger follow-up study is warranted.

Interpretation of serum C-reactive protein (CRP) levels for cardiovascular disease risk is complicated by race, pulmonary disease, body mass index, gender, and osteoarthritis.

OBJECTIVE: High-sensitivity C-reactive protein (hsCRP) in serum is used as a marker of risk for cardiovascular disease (CVD); however CRP is a non-specific acute phase reactant. We evaluated the association between hsCRP concentrations and the most common form of arthritis, osteoarthritis (OA), and assessed the applicability of hsCRP for CVD risk prediction. METHODS: Participants (n=662) were selected from the population-based Johnston County Osteoarthritis Project, using stratified simple random sampling to achieve balance according to radiographic knee OA status, ethnic group, gender, and age group. The presence and severity of knee and hip OA were determined radiographically. CVD risk was estimated by hsCRP concentration and independently with the Framingham risk algorithm. RESULTS: Serum natural log-transformed hsCRP (ln hsCRP) was higher in African-Americans (P<0.0001) and women (P<0.0001), was higher in participants who had chronic pulmonary disease (P=0.01), hypertension (P<0.0001), or used pain medications (P=0.004), and correlated with body mass index (BMI) (r=0.40, P<0.0001) and waist circumference (r=0.33, P<0.0001), but not with age, CVD, or current smoking. Ln hsCRP was strongly positively associated with all definitions of radiographic OA (rOA; P<0.0001), but this association was not independent of BMI. Although 183 participants reported no CVD and were classified as low risk by the Framingham CVD score, 61% of them were classified as moderate or high risk for CVD using hsCRP; this proportion designated high risk for CVD on the basis of hsCRP consisted primarily of women (P<0.05) and individuals with OA (P<0.01). CONCLUSIONS: The pathogenic significance of hsCRP elevations in this subgroup is unclear. Serum hsCRP for predicting risk of CVD is confounded by obesity, ethnicity, gender and comorbidities.

Synovial fluid metabolites in osteonecrosis.

OBJECTIVES: Osteonecrosis of the femoral head results from interruption of the vascular supply and eventual death of the cellular portion of bone. Effective methods of monitoring response to treatment are needed. Our aim was to evaluate synovial fluid metabolites, glucose and lactate, as biomarkers in a canine model of osteonecrosis. METHODS: Osteonecrosis was cryosurgically induced in the right femoral head while the left hip served as control (n = 31). Animals either underwent no further intervention (n = 10), vascular endothelial growth factor (VEGF) injections (n = 4), placement of a vascularized bone graft (n = 6), a combination of VEGF microinjection and vascularized graft placement (n = 5), or treatment with daily oral alendronate (n = 6). After 12 weeks, synovial fluid from each hip joint was obtained for glucose and lactate concentrations. RESULTS: Joints with surgically induced osteonecrosis demonstrated decreased synovial fluid concentrations of glucose (P < 0.05) and elevated concentrations of lactate (P < 0.05) relative to contralateral control hips. When animals were treated with VEGF, the vascularized graft placement, or vascularized graft and VEGF, there were no differences in the synovial fluid concentrations of these metabolites between cryoablated and control hips. In contrast, alendronate did not normalize the concentration of these synovial fluid metabolites in the cryoablated hips. CONCLUSIONS: Osteonecrosis of the femoral head is associated with alterations in synovial fluid glucose and lactate, reflecting anaerobic metabolism. These metabolites may serve as useful tools for monitoring response to revascularization therapies.

The effects of intravenous doxycycline therapy for rheumatoid arthritis: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To determine the feasibility, safety, and potential clinical efficacy of intravenous (IV) doxycycline therapy for rheumatoid arthritis (RA), as well as its possible effects on serum and urinary markers of collagen breakdown. METHODS: The exploratory trial was designed as a 16-week, single-center, randomized, double-blind, placebo-controlled trial. Eligible subjects with active seropositive or erosive RA were randomly allocated into 3 treatment groups: doxycycline 200 mg IV, azithromycin 250 mg orally, or placebo. The blinded IV study drug was administered once daily for the first 3 weeks by home self-infusion and then weekly for the next 8 weeks, concurrent with the blinded oral study drug at the prescribed doses. The primary end points were the change between baseline and week 4 in the tender joint count, erythrocyte sedimentation rate, and urinary excretion of pyridinoline. RESULTS: The trial was stopped prematurely after enrollment of 31 patients. Three subjects were withdrawn because of worsening arthritis, and 1 patient was withdrawn when newly diagnosed with breast cancer. Infusion-related events occurred in 13 (42%) of 31 patients, but none were serious. There were 4 serious adverse events unrelated to the study drug, including a new diagnosis of breast cancer in 3 cases and hospitalization for abdominal pain in 1 case. No significant differences were observed across treatment groups in any of the 3 primary clinical end points. CONCLUSION: Although IV doxycycline therapy was generally well-tolerated by patients in this trial, it did not show any evidence of reducing disease activity or collagen crosslink production.

Comparison of synovial fluid cartilage marker concentrations and chondral damage assessed arthroscopically in acute knee injury.

OBJECTIVE: To examine the correlation between synovial fluid cartilage markers and degree of cartilage damage determined by arthroscopic evaluation in subjects with acute knee injury. DESIGN: Chondral damage was quantified using a validated arthroscopic scoring system in 20 subjects with effusive acute knee injuries of less then 4 months duration and no history or radiographic evidence of joint pathology. Levels of synovial fluid 3B3(-) neoepitope, 3B3(+) chondroitinase generated epitope of proteoglycan, keratan sulfate (KS) and hyaluronic acid (HA) were measured by competitive enzyme-linked immunosorbent assays using monoclonal antibodies 3B3 and 5D4. Total sulfated glycosaminoglycan (GAG) was measured by 1,9-dimethylmethylene blue colorimetric dye-binding assay. RESULTS: We found a dramatic decrease in levels of 3B3(-) (rs = -0.62, P = 0.004), and GAG (rs = -0.49, P = 0.03) with increasing chondral damage score; but no correlation of damage score with 3B3(+), KS or HA levels. CONCLUSION: These data reveal a change in cartilage metabolism within the first 4 months of symptomatic knee injury evinced by a significant inverse correlation of 3B3(-) and GAG levels to chondral lesion severity. These results suggest that serial measurement of these synovial fluid markers in the setting of acute knee injury could predict chondral lesion severity and aid in the decision to intervene surgically.

Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A.

Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex.

E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins.

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.

Promoter-specific trans-activation by the adenovirus E1A12S product involves separate E1A domains.

Recent studies have shown that the adenovirus E1A12S product can trans-activate transcription by activating the transcription factor E2F. However, E2F cannot be the only target for the E1A12S product, since several cellular promoters have been found to be activated by the E1A12S protein even though they lack E2F sites. Indeed, we now show that activation of the hsp70 promoter by the E1A12S product requires the TATAA sequence. Moreover, activation of the hsp70 promoter requires the N-terminal domain of the E1A protein and does not require the conserved region 2 sequences which are required for the E2F-dependent activation of transcription. We conclude that the targeting of distinct transcription factors, leading to trans-activation of transcription of multiple promoters, involves distinct domains of the E1A proteins that are also required for oncogenic activity.

Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product.

The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the transcription factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene.

Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes.

Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of E1A mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.