Background: 3-M syndrome is an autosomal recessive disorder characterized by pre- and post-natal growth restriction, normal intelligence and dysmorphic facial features. Mutations in the genes encoding Cullin 7 (CUL7) and Obscurin like-1 (OBSL1) have been shown to cause 3-M syndrome.
Aims: To characterize CUL7 production and localization in a primary fibroblast cell line from a patient with 3-M syndrome due to a CUL7 mutation and to assess cell proliferation, apoptosis and response to growth hormone (GH) and insulin like growth factor 1 (IGF1) in-patient versus control fibroblasts.
Methods: A dermal fibroblast cell line was established from a patient with a novel nonsense mutation in exon 22 of CUL7 (c.4191delC p.H1379HfsX11). Immunofluorescence using antibodies to CUL7 and the golgi apparatus was carried out in patient and control fibroblasts, with quantitative RT-PCR being used to measure expression of CUL7 and OBSL1. To assess GH and IGF1 signal transduction, control and patient fibroblasts were stimulated with GH and IGF1 with lysates immunoblotted for activation of ERK1/2, STAT5b, IRS-1 and Akt.
Results: The patient fibroblasts demonstrated decreased expression of both CUL7 (relative expression 0.25, P<0.001) and OBSL1 (relative expression 0.45, P<0.001) mRNA compared to control fibroblasts. Western immunoblot demonstrated absence of the 185 kDa band for full length CUL7 with no evidence of truncated protein.
In control fibroblasts CUL7 co-localised with GM130 (a known golgi marker). CUL7 was not identified in the patient fibroblast cell line. Cell proliferation was reduced in patient cells while there was no difference in apoptosis.
Patient fibroblasts displayed normal STAT5b and ERK1/2 signalling following stimulation with rhGH. Activation of IRS1 was normal but reduced levels of activated Akt were seen following IGF1 stimulation.
Conclusions: Loss of CUL7 is associated with reduced OBSL1 transcription, reduced cell proliferation and reduced activation of Akt following stimulation with IGF1.