Hertwig's epithelial root sheath (HERS) is important for tooth root formation, but the molecular basis for the signaling of root development remains uncertain. We hypothesized that Sonic hedgehog (Shh) signaling is involved in the HERS function, because it mediates epithelial-mesenchymal interactions during embryonic odontogenesis. We examined the gene expression patterns of Shh signaling in murine developing molar roots. Shh and Patched2 transcripts were identified in the HERS, whereas Patched1, Smoothened, and Gli1 were expressed in the proliferative dental mesenchyme in addition to the HERS. To confirm whether Shh signaling physiologically functions in vivo, we analyzed mesenchymal dysplasia (mes) mice carrying an abnormal C-terminus of the PATCHED1 protein. In the mutant, cell proliferation was repressed around the HERS at 1 wk. Moreover, the molar eruption was disturbed, and all roots were shorter than those in control littermates at 4 wks. These results indicate that Shh signaling is important in tooth root development. Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; HERS, Hertwig's epithelial root sheath; NFI-C/CTF, nuclear factor Ic/CAAT box transcription factor; PCNA, proliferating cell nuclear antigen; Ptc, patched; Shh, sonic hedgehog; Smo, smoothened.

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Cysteine uptake is the rate-limiting process in glutathione synthesis. Previously we have shown that the inhibitors of excitatory amino acid transporters (EAATs) significantly enhance glutamate toxicity via depletion of intracellular glutathione. In this study we show evidence that the neuronal glutamate transporter EAAT3 is directly enrolled in cysteine uptake in cultured neurons. Neuronal cysteine uptake was dependent on the extracellular sodium, and was suppressed by EAAT inhibitors. Cysteine uptake was suppressed by extracellular glutamate and aspartate, substrates of EAATs, and not by substrates of cysteine transporters. Intracellular glutathione levels were reduced by EAAT inhibitors, and not by inhibitors of cysteine transporters. Knock down of EAAT3 expression using antisense oligonucleotide significantly reduced cysteine uptake, intracellular glutathione level, and neuronal viability against oxidative stress. These facts indicate that EAAT3 functions as a cysteine transporter, and this function seems to be unique and distinct from cysteine transporters that have been reported.

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