We investigated the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 9-cis-retinoic acid (9cRA) on parathyroid hormone-related protein (PTHrP) production and its mRNA expression by the human oral squamous carcinoma cell line (HSC-3). The major transcript of PTHrP was 1.5 kb in human HSC-3 cells. In the presence and absence of serum, 1,25(OH)2D3 produced dose-dependent inhibition of PTHrP gene expression and secretion. Significant inhibition by 1,25(OH)2D3 in the presence of serum was observed at 10(-10) M for mRNA expression and 10(-8) M for secretion, whereas under serum-free conditions, 1,25(OH)2D3 significantly suppressed PTHrP mRNA expression at 10(-10) M and secretion at 10(-9) M. Thus the remainder of the experiments were performed under serum-free conditions. After 24 h of incubation, 9cRA decreased dose-dependently PTHrP mRNA expression and PTHrP secretion. Addition of 10(-7) M 1,25(OH)2D3 or 10(-7) M 9cRA to HSC-3 cells significantly suppressed PTHrp transcription within 1 h and the PTHrP secretion within 12 h. Maximal suppression of mRNA expression was maintained for 12-48 h. 9cRA caused a continuous decrease in PTHrP secretion for up to 48 h, whereas the inhibition of secretion by 1,25(OH)2D3 was transient and abolished by 48 h. Neither 1,25(OH)2D3 nor 9cRA altered the stability of PTHrP mRNA. The inhibitory effect of 1,25(OH)2D3 and 9cRA on PTHrP mRNA expression was additive, whereas no additive effect was observed with regard to PTHrP secretion. These results indicate that 1,25(OH)2D3 and 9cRA suppressed PTHrP production and mRNA expression in oral squamous cancer cells, and suggest that transcriptional suppression may act through binding of the heterodimer (vitamin D receptor-retinoid X receptor) to negatively responsive elements of the PTHrP gene.