Glutamine synthetase is essential for proliferation of fetal skin fibroblasts

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Abstract

Background. Glutamine synthetase (GS) is ubiquitously expressed in the human and plays a major role for many metabolic pathways. However, little is known about its role during the fetal period. Methods. Cultured skin fibroblasts derived from an aborted fetus deficient in GS activity due to a R324C exchange as well as fetal and mature controls were used to determine the level of GS-expression, apoptosis, and proliferation in presence or absence of exogenous glutamine. Results. Glutamine synthetase can be found at early gestational stages. Loss of GS activity either inherited or induced through l-methionine sulfoximine leads to an upregulation of the GS protein but not of the GS mRNA and results in a significant drop in the proliferation rate but has no effect on apoptosis. Exogenous glutamine does not influence the rate of apoptosis but increases proliferation rates of the fetal but not the mature fibroblasts. Conclusion. GS can be found during early human fetal stages when it displays a significant effect on cell proliferation.

Section snippets

Skin fibroblasts

Cultured skin fibroblasts were obtained from an aborted fetus after prenatal diagnosis by molecular genetic means, which had revealed a homozygous mutation (R324C) of the GS gene. The index patient of this family suffered from congenital GS deficiency and has been reported earlier [9]. As a result of the prenatal diagnosis, the gestation was terminated at 16+4 weeks of gestational age and a fetal skin biopsy was taken. The skin biopsy was initially cultured in standard medium containing a

Mutation analysis

Sequencing of the coding exons of the GS gene confirmed the homozygous mutation c.970C>T (R324C) in the fetal GS− skin fibroblasts. Fetal and mature control cells displayed wild type sequences in the GS gene.

Level of GS-expression in cells cultured in standard medium

When fibroblasts were cultured in standard medium containing 2 mM glutamine, Western blot analysis revealed a strong upregulation of GS in the fetal GS− fibroblasts, compared to the GS+ and GS control cells (Fig. 1a).

For analysis of the results of the RT-PCR the calculated relative

Discussion

There is only scarce knowledge on the role of GS during fetal development. We took advantage on the availability of cultured homozygous GS mutant fetal skin fibroblasts and determined their proliferation and viability both in the presence and absence of the GS inhibitor MSO and under the influence of different concentrations of extracellular glutamine.

GS− cells exhibited a significant upregulation of the GS protein, which is in accordance with the findings in immortalized lymphocytes from other

Acknowledgments

The authors are grateful to the technical help of I. Neumann, M. Grüneberg, M. Steinert, T. Janssen, and J. Beckstedde. The work has been supported in part by a grant from the Deutsche Forschungsgemeinschaft to J.H. (HA 4376/1-2) and by the Sonderforschungsbereich 575 “Experimental Hepatology”, Düsseldorf.

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