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A mutation in the c-fos gene associated with congenital generalized lipodystrophy.

Knebel B, Kotzka J, Lehr S, Hartwig S, Avci H, Jacob S, Nitzgen U, Schiller M, März W, Hoffmann MM, Seemanova E, Haas J, Muller-Wieland D - Orphanet J Rare Dis (2013)

Bottom Line: Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity.As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes Research, Duesseldorf, Germany.

ABSTRACT

Background: Congenital generalized lipodystrophy (CGL) or Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.

Methods and results: In a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation (c.-439, T→A) in the patients' c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

Conclusion: The immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development.

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Localization of the postreceptor defect to c-fos expression. A) Western blot analyses of Akt phosphorylation and abundance. B) Phosphorylation of MAPK was assayed by western blot analyses. Activity of MAPK was detected in in-gel kinase assays. C) Formation of the ternary complex at sre element of c-fos promoter in control and patient. The specific complex is indicated by an arrow (lane F: free probe, 1: no competition; competition: 2: 100x SRE, 3: 10x SRE, 4: 100x SP-1, 5: 10x SP-1). D) Activation of ternary complex factor Elk-1 following insulin and PDGF stimulation in patient and control cells. Results are given as means (±S.D.). *p< 0.05 vs basal control. E) Transcriptional activation of c-fos mRNA. The mRNA levels were normalized against 18S rRNA as internal control. Values are means (±S.D.) from four independent experiments, each performed in triplicate. *p< 0.05 vs basal control.
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Figure 1: Localization of the postreceptor defect to c-fos expression. A) Western blot analyses of Akt phosphorylation and abundance. B) Phosphorylation of MAPK was assayed by western blot analyses. Activity of MAPK was detected in in-gel kinase assays. C) Formation of the ternary complex at sre element of c-fos promoter in control and patient. The specific complex is indicated by an arrow (lane F: free probe, 1: no competition; competition: 2: 100x SRE, 3: 10x SRE, 4: 100x SP-1, 5: 10x SP-1). D) Activation of ternary complex factor Elk-1 following insulin and PDGF stimulation in patient and control cells. Results are given as means (±S.D.). *p< 0.05 vs basal control. E) Transcriptional activation of c-fos mRNA. The mRNA levels were normalized against 18S rRNA as internal control. Values are means (±S.D.) from four independent experiments, each performed in triplicate. *p< 0.05 vs basal control.

Mentions: The in vitro insulin receptor binding capacity, auto- and substrate-phosphorylation was in normal range (data not shown). The insulin mediated signaling cascade including Akt abundance or phosphorylation (Figure 1A) and ERK1/2-MAPK activation or activity (Figure 1B) was comparable to controls in patient cells, indicating no disturbance in these pathways. A definite endpoint of MAPK cascades is the transcriptional activation of the immediate-early genes c-fos [17], whereas the c-fos gene activity is directly related to formation of a ternary transcription activation complex at the sre-element and the phosphorylation of the ternary complex factor Elk-1 by ERK1/2-MAPK [18]. Investigations showed that the formation of the ternary complex (Figure 1C) and Elk-1-activation dependent transcription was not altered in cells of the patient (Figure 1D). Nevertheless in patient cells induction of c-fos mRNA expression was nearly completely lost following insulin and IGF-1 induction and clearly reduced following PDGF and EGF induction in contrast to control cells (Figure 1E). These findings focused the defect to c-fos gene directly.


A mutation in the c-fos gene associated with congenital generalized lipodystrophy.

Knebel B, Kotzka J, Lehr S, Hartwig S, Avci H, Jacob S, Nitzgen U, Schiller M, März W, Hoffmann MM, Seemanova E, Haas J, Muller-Wieland D - Orphanet J Rare Dis (2013)

Localization of the postreceptor defect to c-fos expression. A) Western blot analyses of Akt phosphorylation and abundance. B) Phosphorylation of MAPK was assayed by western blot analyses. Activity of MAPK was detected in in-gel kinase assays. C) Formation of the ternary complex at sre element of c-fos promoter in control and patient. The specific complex is indicated by an arrow (lane F: free probe, 1: no competition; competition: 2: 100x SRE, 3: 10x SRE, 4: 100x SP-1, 5: 10x SP-1). D) Activation of ternary complex factor Elk-1 following insulin and PDGF stimulation in patient and control cells. Results are given as means (±S.D.). *p< 0.05 vs basal control. E) Transcriptional activation of c-fos mRNA. The mRNA levels were normalized against 18S rRNA as internal control. Values are means (±S.D.) from four independent experiments, each performed in triplicate. *p< 0.05 vs basal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750569&req=5

Figure 1: Localization of the postreceptor defect to c-fos expression. A) Western blot analyses of Akt phosphorylation and abundance. B) Phosphorylation of MAPK was assayed by western blot analyses. Activity of MAPK was detected in in-gel kinase assays. C) Formation of the ternary complex at sre element of c-fos promoter in control and patient. The specific complex is indicated by an arrow (lane F: free probe, 1: no competition; competition: 2: 100x SRE, 3: 10x SRE, 4: 100x SP-1, 5: 10x SP-1). D) Activation of ternary complex factor Elk-1 following insulin and PDGF stimulation in patient and control cells. Results are given as means (±S.D.). *p< 0.05 vs basal control. E) Transcriptional activation of c-fos mRNA. The mRNA levels were normalized against 18S rRNA as internal control. Values are means (±S.D.) from four independent experiments, each performed in triplicate. *p< 0.05 vs basal control.
Mentions: The in vitro insulin receptor binding capacity, auto- and substrate-phosphorylation was in normal range (data not shown). The insulin mediated signaling cascade including Akt abundance or phosphorylation (Figure 1A) and ERK1/2-MAPK activation or activity (Figure 1B) was comparable to controls in patient cells, indicating no disturbance in these pathways. A definite endpoint of MAPK cascades is the transcriptional activation of the immediate-early genes c-fos [17], whereas the c-fos gene activity is directly related to formation of a ternary transcription activation complex at the sre-element and the phosphorylation of the ternary complex factor Elk-1 by ERK1/2-MAPK [18]. Investigations showed that the formation of the ternary complex (Figure 1C) and Elk-1-activation dependent transcription was not altered in cells of the patient (Figure 1D). Nevertheless in patient cells induction of c-fos mRNA expression was nearly completely lost following insulin and IGF-1 induction and clearly reduced following PDGF and EGF induction in contrast to control cells (Figure 1E). These findings focused the defect to c-fos gene directly.

Bottom Line: Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity.As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes Research, Duesseldorf, Germany.

ABSTRACT

Background: Congenital generalized lipodystrophy (CGL) or Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.

Methods and results: In a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation (c.-439, T→A) in the patients' c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

Conclusion: The immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development.

Show MeSH
Related in: MedlinePlus