Figure 4

Papss2 is regulated by transforming growth factor β. (A) RNA isolated from bovine chondrocytes grown in micromass cultures and either left untreated or treated with 5 ng/transforming growth factor β (TGF-β)/ml for 8 hours (n = 3 separate cows) was used in real-time RT-PCR. Data are shown as tables obtained using REST software [30]. Papss2 and two other selected genes (Prg4 and Plod2) were upregulated after treatment with TGF-β, confirming the microarray results. Gapdh was used as a normalization control. (B) Gene expression was compared by real-time RT-PCR using RNA isolated from 20-week-old wild-type (WT) and dominant-negative TGF-β type II receptor (DNIIR) mice (n = 5 controls and 6 mutants) using REST software. Significant downregulation of Prg4, Papss2 and Plod2 was seen in DNIIR samples. Hprt was used as a normalization control. Fxyd2 was not regulated by TGF-β on the microarray, which we verified by RT-PCR. (C) Cell protein lysates were isolated from articular cartilage of 6-week-old, 20-week-old and 10-month-old control and DNIIR mice (n = 3 each at age 6 weeks, 4 each at age at 20 weeks and 2 each at age 10 months). Papss2 expression was determined by Western blot analysis. Gapdh was used as a loading control. The average Papss2/Gapdh ratios derived from all of the blots are shown, with the ratio for WT set to 1. Papss2 protein was reduced in DNIIR mice at each stage.