Tomoko Hiroi

Suppressive effects of tranilast on the expression of inducible cyclooxygenase (COX2) in interleukin-1β-stimulated fibroblasts

We investigated the effects of tranilast on inducible cyclooxygenase (COX2)-mediated prostaglandin E2 (PGE2) production and enzyme induction in interleukin-lbeta (IL-1beta)-stimulated cultured dermal fibroblasts. IL-1beta enhanced PGE2 production in cultured fibroblasts. Tranilast did not affect constitutive cyclooxygenase (COX1) or COX2 activity in non-stimulated or IL-lbeta-stimulated fibroblasts. However, the COX2 expression induced by IL-1beta was inhibited by tranilast. This result, that IL-1beta-induced COX2 expression was suppressed by tranilast, was confirmed by immunohistochemical analysis. Thus, it is possible for tranilast to regulate PGE2 production by inhibiting COX2 induction.

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5′-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5′-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly67–Leu68 or Leu68–Trp69.

Functional characterization of the neuron-restrictive silencer element in the human tryptophan hydroxylase 2 gene expression

Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 neuron‐restrictive silencer element (NRSE) functions as a negative regulator dependent on neuron‐restrictive silencer factor (NRSF) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE‐mediated repression of the human TPH2 (hTPH2) promoter activity in RN46A cells, a cell line derived from rat raphe neurons. Quantitative real‐time RT‐PCR analysis revealed the expression of serotonergic marker genes (Mash1, Nkx2.2, Gata2, Gata3, Lmx1b, Pet‐1, 5‐Htt, and Vmat2) and Nrsf gene in RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF‐dependent repression of the hTPH2 promoter activity. The hTPH2 promoter activity was increased by knockdown of NRSF, or over‐expression of the engineered NRSF (a dominant‐negative mutant or a DNA‐binding domain and activation domain fusion protein). MS‐275, a class I histone deacetylase (HDAC) inhibitor, was found to be more potent than MC‐1568, a class II HDAC inhibitor, in enhancing the hTPH2 promoter activity. Furthermore, treatment with the ubiquitin‐specific protease 7 deubiquitinase inhibitors, P‐22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE‐mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin‐specific protease 7‐mediated deubiquitination and stabilization of NRSF.