The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts

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Abstract

Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10-7, 10-6, 10-5 M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10-6 M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription on bone-type mRNA of the TNSALP gene.

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