Serine hydroxymethyltransferase (SHMT) is the primary enzyme in the interconversion of serine and glycine. The roles of mitochondrial and cytosolic SHMT in the interconversion of serine and glycine were determined in two Chinese hamster ovary (CHO) cell lines that both contain cytosolic SHMT but either have (CHOm+) or lacK (CHOm-) mitochondrial SHMT. Mitochondrial SHMT activity was significantly reduced in CHOm- (0.24 +/- 0.11 nmol/min per mg of mitochondrial protein) compared with CHOm+ (3.21 +/- 0.66 nmol/min per mg of mitochondrial protein; P = 0.02) cells, whereas cytosolic SHMT activity was similar in CHOm- and CHOm+ cells (1.09 +/- 0.31 and 1.53 +/- 0.12 nmol/min per mg of cytosolic protein respectively; P = 0.57). In CHOm+ and CHOm- cells, the relative flux of glycine to serine measured with either [1-13C]- or [2-13C]-glycine was similar (CHOm-: 538 +/- 82 nmol/24 per mg of DNA; CHOm+: 616 +/- 88 nmol/24 h per mg of DNA; P = 0.42). In contrast, the relative flux of serine to glycine measured with [1-13C]serine was low in CHOm- cells (80 +/- 28 nmol/24 h per mg of DNA) compared with CHOm+ cells (3080 +/- 320 nmol/24 h per mg of DNA; P = 0.0001). The rate of glycine production determined by [1-(13)C]glycine dilution was lower in CHOm- (1200 +/- 200 nmol/24 h per mg of DNA) than CHOm+ (10200 +/- 1800 nmol/24 h per mg of DNA; P = 0.03) cells, whereas glycine utilization was similar in the two cell lines. Serine production was similar in the two cell lines but serine utilization was lower in CHOm- (3800 +/- 1200 mu mol/24 h per mg of DNA) than CHOm+ (6600 +/- 1000 nmol/24 h per mg of DNA; P = 0.0002) cells. Increasing the serine concentration in the medium resulted in an increase in glycine production in CHOm+ but not in CHOm- cells. Intracellular studies with [1-13C]serine confirm the findings of decreased glycine production from serine. In CHO cells there is partitioning of intracellular serine and glycine metabolism. Our data support the hypothesis that mitochondrial SHMT is the primary pathway for serine into glycine interconversion.

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We have previously shown that the estrogen responsiveness of the human lactoferrin gene in a transient transfection system is mediated through an imperfect estrogen response element (ERE) and a steroidogenic factor 1 binding element (SFRE) 26 bp upstream from ERE. Reporter constructs containing SFRE and ERE respond to estrogen stimulation in a dose-dependent manner, whereas mutations at either one of the response elements severely impaired the estrogen responsiveness. In this study, we demonstrated that estrogen receptor (ERalpha) binds to the human lactoferrin gene ERE and forms two complexes in an electrophoresis mobility shift assay (EMSA). These complexes could be supershifted by an antibody to ERalpha. We also showed that in normal cycling women, lactoferrin gene expression in the endometrium increases during the proliferative phase and diminishes during the luteal phase. This in-vivo study thus supported the finding from transient transfection experiments that the human lactoferrin gene expression is elevated in an environment with a high level of estrogen. The estrogen effect on lactoferrin gene expression in the rhesus monkey endometrium was studied by Western blotting and immunohistochemistry. The immunohistochemistry results showed that immunoreactive lactoferrin protein was not detectable in the untreated ovariectomized monkey endometrium, was elevated by estrogen treatment, and was suppressed by sequential, combined estrogen plus progesterone treatment. In conclusion, this study has shown that lactoferrin gene expression is responsive to estrogen in primate endometrium.

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