SOX9 protein is stabilized by TGF-ß and regulates PAPSS2 mRNA expression in chondrocytes.

OBJECTIVE: We previously identified 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) as a transcriptional target of transforming growth factor ß (TGF-ß) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-ß responsive gene, proteoglycan 4/lubricin (PRG4). In this study, TGF-ß-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. DESIGN: Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3. Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. RESULTS: Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-ß treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-ß signaling. Furthermore, Sox9 was required for full TGF-ß-mediated induction of Papss2. In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-ß, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-ß. CONCLUSIONS: TGF-ß stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-ß-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-ß-mediated gene regulation in chondrocytes.

Diagnostic usefulness of lung SPET in pulmonary thromboembolism: an outcome study.

The lung single photon emission tomographic (SPET) images of 985 consecutive patients referred for suspected pulmonary embolism were correlated with clinical outcome and angiography to evaluate the clinical usefulness of lung SPET compared to conventional planar ventilation/perfusion lung imaging. SPET interpretations followed the revised PIOPED criteria and clinical outcome was determined from referring physicians, hospital records, direct patient contact and county hall records. Patients were deemed to have had no clinically significant pulmonary embolism at the time of the SPET examination if, within the following 3 months: (1) the patient was alive and had no clinical evidence of pulmonary embolism or, (2) if deceased, pulmonary embolism was unlikely to have been the cause of death. Operating characteristics were based on the methods of Choi and of Simel. SPET interpretation was categorized as follows: high probability, 143 (14%); low probability, 840 (82%); intermediate, 41 (4%) (in contrast to PIOPED, with 39% intermediate interpretations). Pulmonary angiography was performed in only 4% of patients. Adequate follow-up data were available for 97% of patients. To facilitate comparison with PIOPED, either a high-probability or an intermediate-probability or an intermediate-probability study was considered to be a positive test, and either a low-probability or a normal study was considered to be a negative test. The sensitivity was 83% (PIOPED 82%), specificity 92% (PIOPED 52%), positive predictive value 62% (PIOPED 47%) and negative predictive value 97% (PIOPED 85%). The positive and negative predictive values have not been corrected for prevalence, which was approximately twice as high in the PIOPED study. Lung SPET provided accurate diagnostic information in 96% of patients and specificity was greatly improved compared to planar lung imaging reported in PIOPED. The diminished need for angiography greatly reduced the cost of evaluating patients suspected of having pulmonary embolism.