Stimulation of tryptophan hydroxylase production in a serotonin‐producing cell line (RBL2H3) by intracellular calcium mobilizing reagents

Hasegawa, Hiroyuki; Kojima, Masayo; Iida, Yasunori; Oguro, Kazuya; Nakanishi, Nobuo

doi:10.1016/0014-5793(96)00834-4

Cited by 10 publications

(9 citation statements)

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“…On the other hand, RBL-2H3 cells are basophilic leukemia cells and have been extensively studied for their ability to produce serotonin. These cells have been shown to contain serotonin and TPH (Hasegawa et al, 1996) and to transport serotonin across the cell membrane (Kanner and Bendahan, 1985). Thus, although RBL-2H3 is not of neuronal origin, it is currently the optimal in vitro model system with which to study the serotonin synthesis pathway by biochemical and molecular biological techniques.…”

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Up-Regulation of Tryptophan Hydroxylase Expression and Serotonin Synthesis by Sertraline

2002

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The neurotransmitter serotonin is involved in a variety of brain functions, and abnormal changes in serotonin neurotransmission are associated with an array of psychiatric disorders, including depression. Sertraline is a selective serotonin reuptake inhibitor (SSRI) and an effective antidepressant. Sertraline increases the serotonin concentration in the synaptic cleft by a short-term action; however, clinical improvement is observed only after several weeks, suggesting that the therapeutic effect may be caused by long-term alterations in serotonin transmission. We determined the effects of sertraline on serotonin synthesis in vivo and in vitro. Long-term treatment of rats with sertraline up-regulated mRNA and protein levels of the serotonin-synthesizing enzyme tryptophan hydroxylase (TPH), as determined by in situ hybridization and immunocytochemistry, respectively. In vitro studies using RBL-2H3 cells also showed an increase in mRNA and protein levels of TPH by sertraline, as determined by Northern blot and immunoblot analyses, respectively. This was accompanied by increases in the levels of TPH enzymatic activity and total serotonin. These data demonstrate that in addition to the known short-term action as an uptake blocker, sertraline also exerts a long-term effect on the serotonin neurotransmission by enhancing serotonin synthesis. A similar effect was observed with another SSRI, fluoxetine, but not with the non-SSRI chlorpromazine. The up-regulation of TPH gene expression by sertraline was attenuated by the protein kinase A (PKA) inhibitor N-[2-(p-bromocinnamylamine)-ethyl]-5-isoquinolinesulfonamine, suggesting that a mechanism involving the PKA signaling pathway might at least in part mediate the long-term therapeutic action.

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Up-Regulation of Tryptophan Hydroxylase Expression and Serotonin Synthesis by Sertraline

2002

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“…Complimentary DNAs of TPH have been cloned from various sources but no differences or only trivial variation in amino acid sequences were found among them [14][15][16][17][18][19]. The molecular basis of differences between the neural and non-neural enzymes has not yet been explained.Both types of cytosolic environment should be studied further to detect differences in the control of gene expression, post-translational modification, and turnover of the enzyme protein in a tissue-specific way.We have demonstrated with RBL2H3, an established cell line that expresses TPH in culture while retaining many of the characteristics of mast cells, that: (a) cellular TPH activity was seriously limited by insufficient supply with the enzyme's essential cofactor, ferrous iron, and the substrates tryptophan and 6R-tetrahydrobiopterin [20]; (b) immune stimulation lead to a marked increase in TPH level by means of enhanced expression of the TPH gene [21]; and (c) the steady state TPH level of this cell was maintained at extremely low levels by rapid degradation of the enzyme (T 1/2 , 15-60 min) [22,23]. In the latter report, the turnover of TPH protein was shown to be driven by ATP-dependent action of 26S-proteasomes including, at least in part, ubiquitinylation of TPH.…”

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Proteasome‐driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line

Iida

Sawabe

Kojima

et al. 2002

European Journal of Biochemistry

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We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127,[121][122][123][124][125][126][127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32 P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca 2+ /calmodulindependent protein kinase II in the presence of Ca 2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca 2+ /calmodulindependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation.Keywords: tetrahydrobiopterin; CaM kinase II; proteasome target; ubiquitin ligase; enzyme turnover.Tryptophan hydroxylase (TPH, EC 1.14.16.4), a member of a family of pterin-dependent aromatic amino acid hydroxylases [1], catalyzes the conversion of L-tryptophan to 5-hydroxy-L-tryptophan. This reaction is the initial and rate-limiting step in the biosynthesis of serotonin [2][3][4][5]. TPH has been extensively purified from various sources such as bovine pineal gland [6], mouse mastocytoma [7,8], and mammalian brains [9][10][11]. Physicochemical, enzymic and immunochemical properties differed between TPHs of neural and non-neural tissue origin, and it is accepted that neural TPH might be a different entity from the non-neural enzyme [8,10,12,13]. Complimentary DNAs of TPH have been cloned from various sources but no differences or only trivial variation in amino acid sequences were found among them [14][15][16][17][18][19]. The molecular basis of differences between the neural and non-neural enzymes has not yet been explained.Both types of cytosolic environment should be studied further to detect differences in the control of gene expression, post-translational modification, and turnover of the enzyme protein in a tissue-specific way.We have demonstrated with RBL2H3, an established cell line that expresses TPH in culture while retaining many of the characteristics of mast cells, that: (a) cellular TPH activity was ...

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“…Biological properties of TPH have been extensively investigated through purification of this enzyme from a variety of samples such as mammalian brain (Cash et al, 1985;Ichiyama et al, 1970;Fujisawa, 1981, 1982a), murine small intestine Noguchi et al, 1973), neoplastic murine mast cells (Hasegawa et al, 1996;Hosoda andGlick, 1965, 1966;Hosoda, 1975;Nakata and Fujisawa, 1982b;Yanagisawa et al, 1982), and human carcinoid tumor (Grahame-Smith, 1964;Hosoda et al, 1977). The half-life of this enzyme is very short (Hasegawa et al, 1995(Hasegawa et al, , 1996, and its highly unstable nature has been an obstacle to producing a specific antibody against TPH.…”

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“…The half-life of this enzyme is very short (Hasegawa et al, 1995(Hasegawa et al, , 1996, and its highly unstable nature has been an obstacle to producing a specific antibody against TPH. Several investigators have attempted to develop the specific antisera against TPH purified from rat brain (Cash et al, 1985;Weissmann et al, 1987) and from mouse mastocytoma Nakata and Fujisawa, 1982b).…”

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Immunohistochemical localization of tryptophan hydroxylase in the human and rat gastrointestinal tracts

Fujimura

Okumiya

et al. 1999

J. Comp. Neurol.

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Stimulation of tryptophan hydroxylase production in a serotonin‐producing cell line (RBL2H3) by intracellular calcium mobilizing reagents

Cited by 10 publications

References 24 publications

Up-Regulation of Tryptophan Hydroxylase Expression and Serotonin Synthesis by Sertraline

Up-Regulation of Tryptophan Hydroxylase Expression and Serotonin Synthesis by Sertraline

Proteasome‐driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line

Immunohistochemical localization of tryptophan hydroxylase in the human and rat gastrointestinal tracts

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