J Pediatr Endocr Met 2012;25(3-4):273–278 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/jpem-2011-0491
Expression of SOCS1, SOCS2, and SOCS3 in growth hormone-stimulated skin fibroblasts from children with idiopathic short stature1)
Paula Ocaranza*, Fernanda Morales, Rossana Román, Germán Iñiguez and Fernando Cassorla Institute of Maternal and Child Research, School of Medicine, University of Chile, Santiago, Chile
Abstract Background/aim: Possible etiologies of idiopathic short stature (ISS) include a range of conditions, some of which may be caused by defects in the modulation of the growth hormone (GH)-signaling pathway. The Janus kinase/ signal transducer and activator of transcription pathway is regulated by several mechanisms, including negative feedback regulation by the suppressors of cytokine signaling (SOCS). However, the specific induction of SOCS transcript levels in fibroblasts from ISS patients has not been studied. Methods: We determined the transcript levels of the SOCS1–3 genes under basal conditions, and in the presence or absence of stimulation with rhGH for 24 h in skin fibroblast cultures obtained from patients with ISS and children with normal height. Results: Under basal conditions, ISS patients express higher SOCS2–3 transcript levels than control children. After incubation with recombinant human GH (rhGH), the transcript levels of SOCS2 increased significantly in ISS patients compared to controls (0.79 ± 0.06 vs. 0.55 ± 0.07; p = 0.03), a pattern which did not achieve statistical significance for SOCS3 transcript levels (0.55 ± 0.08 vs. 0.40 ± 0.07). Conclusion: The higher baseline transcript levels of the SOCS genes, and the increase observed for SOCS2 after rhGH treatment in ISS patients, suggest that growth retardation in some of these children may be mediated, at least in part, by intracellular overexpression of the SOCS genes. Keywords: growth hormone; idiopathic short stature; signal transduction; suppressors of cytokine signaling (SOCS). 1) Supported in part by FONDECYT grant 1095118 and PBCT PSD51. *Corresponding author: Paula Ocaranza, PhD, Institute of Maternal and Child Research, School of Medicine, University of Chile, Avenida Santa Rosa 1234, piso 2, PO Box 226-3, Santiago, 8360160, Chile Phone: +56 (2) 977 0851, Fax: +56 (2) 424 7240, E-mail:
[email protected] Received December 23, 2011; accepted January 13, 2012; previously published online February 27, 2012
Introduction The term idiopathic short stature (ISS) is applied to a broad range of short children with a variety of conditions, such as those with familial short stature, constitutional growth delay, and those who are abnormally short for their parental target height, some of whom may harbor an unrecognized endocrine defect (1, 2). The syndrome of growth hormone (GH) insensitivity has been described in patients with increased concentrations of GH and decreased levels of insulin-like growth factor-I (IGF-I) (1). This condition may be caused by different factors affecting the GH signaling transduction pathway. A related syndrome of partial GH insensitivity has been observed among some children with suspected ISS, who have relatively low levels of IGF- I and relatively high levels of GH (3); however, the specific cause of this partial GH insensitivity has not been defined in most cases. The signal transduction cascade induced by the GH receptor (GHR) triggers a complex array of biochemical events acting in a coordinated fashion and involves a large number of distinct molecules. GH interaction to its membrane receptor and induced receptor dimerization activates the Janus kinase (JAK2) (4), which in turn induces tyrosine phosphorylation within itself and the GHR. These tyrosines form binding sites for a number of signaling proteins, including members of the family of signal transducers and activators of transcription (STATs) (5, 6), specifically STAT1, STAT3, STAT5a, and STAT5b. STAT dimers translocate into the nucleus, where they bind to specific DNA elements and initiate gene transcription (2, 3, 6, 7). The suppressors of cytokine signaling (SOCS) proteins are generated in response to cytokines and can also bind to phosphorylated tyrosines within the GHR-JAK complex, and further inhibit cytokine receptor activation (8). The SOCS family consists of at least eight members: SOCS1–7 and the cytokine inducible SH-2 containing protein (CIS) (9, 10). These proteins regulate in a negative fashion the strength and duration of the signaling cascade, particularly that which utilizes the JAK/STAT systems, such as those induced by insulin and GH (11–13). The model of SOCS action is that of a classic feedback loop, whereby factor-induced activation of signaling pathways induces expression of effector genes, including those encoding the SOCS proteins. These proteins attenuate the signal by inhibiting various components of the pathway (14). Direct interaction of SOCS with the JAK kinases or cytokine receptors allows their recruitment to the signaling complex
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Ocaranza et al.: Expression of SOCS in children with idiopathic short stature
where they inhibit JAK catalytic activity, or block access of the STATs to the receptor binding sites (15, 16). The aim of this study was to investigate the intracellular gene expression of SOCS1, SOCS2, and SOCS3 in children with ISS compared to controls, in an effort to document whether these negative regulators of GH signaling are overexpressed in patients with ISS. To test this hypothesis, we determined the expression of these transcript levels in skin fibroblasts from children with ISS and apparent low GH sensitivity and control children with normal height. The transcript levels in ISS and control children were assayed under basal conditions and in the presence or absence of rhGH stimulation.
(Webster, TX, USA). The sensitivity of this assay is 0.1 mg/L, with intra- and interassay CVs of 1.1% and 1.8%, respectively.
Cell culture Primary fibroblast cultures were established from skin-biopsy specimens obtained from the patients (internal elbow fold) and healthy control children (at the time of surgery). This tissue (∼1 mm3) was cultured in DMEM supplemented with 10% FCS, 10 U/mL, penicillin/streptomycin, and 0.25 μg/mL fungizone at 37°C in a 5% CO2 atmosphere. Fibroblast cultures were used between the third and sixth passage to avoid the influence of plasma factors and senescent changes in the cellular response.
Analysis of SOCS1, SOCS2, and SOCS3 in skin fibroblasts by reverse transcription-PCR
Materials and methods Study subjects The study population consisted of 11 prepubertal children (7.3 ± 0.5 years of age) with ISS who met the following inclusion criteria: (i) height < 2 standard deviation score (SDS) and growth velocity under the 10th percentile for age and sex, (ii) a GH response to a clonidine stimulation test > 15 ng/mL, (iii) serum IGF-I or IGF binding protein (IGFBP)-3 levels below the mean for age and sex, and (iv) without apparent mutations in the GHR. The control group included 11 prepubertal healthy children (6.3 ± 0.4 years of age) with normal stature and weight for age and sex who underwent elective surgery for an unrelated condition. The subjects were recruited at the San Borja-Arriarán Hospital, and the study was approved by the Ethics Committee of the University of Chile. Informed consent was obtained from the parents of the patients and the control children enrolled in the study.
Chemicals Recombinant human GH (rhGH) was a gift from Dr. A.F. Parlow (National Hormone and Peptide Program, NIH, USA). Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L of glucose, Dulbecco’s phosphate buffered solution (DPBS), penicillin/streptomycin, and fungizone were purchased from Invitrogen (Grand Island, NY, USA). Heat inactivated fetal calf serum (FCS) was from Biological Industries (Kibbutz Beit Haemek, Israel). Prestained molecular mass standard was from Fermentas (Ontario, Canada), and the other chemicals were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise.
Assays Serum GH was measured by a double antibody radioimmunoassay (RIA) with 0.8 ng/mL sensitivity, and intra- and interassay coefficients of variation (CVs) of 10% and 6.5%, respectively. The GH values were calibrated against the standard reference WHO 1st IS 88/624. All reagents for the GH RIA (human GH-I-3, antihuman GH-2 antisera, human GH-RP) were donated by the National Hormone and Pituitary Program. Serum IGF-I levels were determined by RIA after an ethanol-acid sample extraction as a first step to remove the IGFBPs, as previously described by Iñiguez et al. (17). The sensitivity of this assay is 5 ng/mL, and the intra- and interassay CVs are 8.6% and 10.2%, respectively. Serum IGFBP-3 levels were measured by an immunoradiometric assay from Diagnostic System Laboratories
Skin fibroblasts seeded on 100-mm dishes at 80%–90% confluence were washed with DPBS and serum deprived for 48 h. Two days after deprivation, the cells were treated with or without 200 ng/mL rhGH for 24 h. Total RNA was extracted with TRIzol reagent (Invitrogen, Bethesda, MD, USA) from treated skin fibroblast cell cultures and skin fibroblast cultures under basal conditions. Reverse transcription of 3 μg of total RNA was performed in 20 μL reactions containing 1 μL/ reaction of Impron-II reverse transcriptase (Promega, Madison, WI, USA), 50 mM Tris-HCl (pH 8.3, 25°C), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM of each deoxyribonucleotide triphosphate, and 0.5 μg of random primers. Reactions were conducted at 39°C for 60 min. Two microliters of this reaction was used for PCR amplification in a 20 μL reaction containing 2.0 mM MgCl2, 0.5 mM each deoxyribonucleotide triphosphate, 2.5 U of GoTaq DNA polymerase (Promega), 10 mM Tris-HCl (pH 8.5, 25°C), 50 mM KCl, 0.1% Triton X-100, and 25 pmol of each primer. The PCR program and SOCS primers were described by Tsao et al. (18), and consisted of an initial cycle of denaturation (2 min, 94°C) followed by 33 cycles of denaturation (1 min, 94°C), annealing (1 min, 59°C), and extension (1 min, 72°C). Primers for SOCS1: 5′-GAACTGCTTTTTCGCCCTTAG-3′ (forward) and 5′-TCAAATCTGGAAGGGGAAGGA-3′ (reverse) yield a 310-bp product; SOCS2: 5′-TTTGGGATTCGCACTGACTTC-3′ (forward) and 5′-GTTCCTTCTGGTGCCTCTTTT-3′ (reverse) yield a 300-bp product; SOCS3: 5′-TTCTTCACGCTCAGCGTCAAG-3′ (forward) and 5′-ATGTAATAGGCTCTTCTGGGG-3′ (reverse) yield a 266-bp product (18). For normalization purposes, the β-actin cDNA was also measured using the primers 5′-GGCCGGGACCTGACTGACTA-3′ (forward) and 5′- GGAAGGAAGGCTGGAAGAG-3′ (reverse) and yield a 256-bp product. DNA fragments in PCR products were separated on 2% agarose gels, followed by ethidium bromide staining to visualize DNA fragments in gels. The signal intensity of DNA fragments in PCR products was quantified by using ImageJ 1.43x (NIH, Bethesda, MD, USA).
Statistical analyses The SDS for body mass index (BMI), weight, and height for our patients were based on the National Center for Health Statistics. Results are shown as mean±SEM. Differences between groups were determined by the Student’s t-test for parametric variables, and by the Mann-Whitney test for non-parametric variables. The differences between basal and 24 h with and without rhGH for each subject were compared by the Wilcoxon test. Statistical analysis was performed with the IBM-SPSS program v11.5 (New York, USA), and p-values
