Alterations of a serum marker of collagen X in growing children with osteogenesis imperfecta.

Abnormalities in the structure and/or processing of type I collagen cause osteogenesis imperfecta and result in bone fragility, abnormal bone growth and short stature. Type I collagen is expressed in the growth plate but the mechanisms by which abnormalities in collagen I contribute to growth plate dysfunction and growth retardation are unknown. The non-collagenous domain (NC1) of type X collagen (CXM) is released from the hypertrophic zone of active growth plates and is a marker for new endochondral bone formation. Serum CXM levels are strongly correlated with the rate of growth in healthy children. We hypothesized that CXM levels in children with OI would be abnormal when compared to normally growing children. Using participants from the Brittle Bone Disease Consortium Natural History Study we analyzed the distribution of CXM over the ages of 8 months to 40 years in 187 subjects with OI (89 type I and 98 types III/IV) as well as analyzed the relationship between growth velocity and CXM levels in a subset of 100 children <16 years old with OI (44 type I and 56 types III/IV). CXM levels in both control and OI children demonstrated a similar pattern of variation by age with higher levels in early life and puberty followed by a post-pubertal drop. However, there was greater variability within the OI cohort and the relationship with growth velocity was weaker. The ratio of CXM level to growth velocity was elevated in children with type III/IV OI compared to controls. These results suggest that the relationship between hypertrophic zone function and the end point of skeletal growth is disrupted in OI.

Impact of age, phenotype and cardio-renal function on plasma C-type and B-type natriuretic peptide forms in an adult population.

CONTEXT: In contrast to the cardiac hormones, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), variations in plasma concentrations of C-type natriuretic peptide (CNP) in healthy adults are ill-defined, limiting their clinical application. OBJECTIVE: Our objective was to define the effect of age, phenotype (gender, height, BMI), and cardiac and renal function on plasma CNPs in an adults population without renal or cardiovascular disease. DESIGN AND SETTING: This was a prospective cross-sectional observational study of adult volunteers, aged 21-80 years, randomly selected from the electoral roll. SUBJECTS AND METHODS: Plasma CNP and its associated aminoterminal propeptide (NTproCNP) were measured in 258 subjects and related to age, gender, height and plasma creatinine. Subgroup analyses seeking associations with cardiac function (plasma BNP and NTproBNP) and bone turnover bone-specific alkaline phosphatase (bALP) were also determined. RESULTS: Plasma concentrations of CNPs in men continued to decline from adolescent values to reach a nadir in the 5th decade after which values increased. Similar but less marked changes occurred in women. In both sexes, NTproCNP was inversely and independently correlated with height. In contrast to B-type natriuretic peptides (BNPs), NTproCNP was higher in men, significantly related to creatinine and positively related to bALP. CONCLUSIONS: Gender- and age-specific changes affect CNPs in adults. Inverse associations of NTproCNP with adult height, positive correlation with creatinine - and in contrast to CNP - no association with BNP are further unique findings distinguishing NTproCNP, which need to be considered in future studies.

Plasma C-type natriuretic peptide forms and thyroid status in prepubertal children with acquired thyroid disease.

OBJECTIVE: C-type natriuretic peptide (CNP) and thyroid hormone (TH) are essential for normal skeletal growth. Plasma CNP peptides correlate with growth velocity, but the relationship between thyroid status and CNP production is unknown. This study examined the impact of restoring normal TH levels on CNP and height velocity (HV) in children with acquired hypo- and hyperthyroidism. DESIGN: We performed a prospective, observational study in prepubertal children with acquired hypothyroidism (n = 15) and hyperthyroidism (n = 12). MEASUREMENTS: Blood levels of CNP, amino-terminal proCNP (NTproCNP), bone-specific alkaline phosphatase (BSAP), IGF-I and TH levels were measured before and during the first 6 months of standard treatment for hypo- and hyperthyroidism, and correlations were determined. RESULTS: At baseline, HV, CNP, NTproCNP and BSAP were significantly higher in hyper- than in hypothyroid subjects. Changes in TH after treatment were closely coupled to change in CNP and NTproCNP in hyperthyroid, but not in hypothyroid, children. In addition, a positive association of HV with CNP peptides was found during treatment of hyperthyroidism. Normalizing TH did not correlate with changes in BSAP or IGF-I in either group. CONCLUSIONS: Plasma CNP peptides are higher in children with hyperthyroidism than in those with hypothyroidism at diagnosis and, in hyperthyroid children, change concordantly with TH and HV during treatment. Differential responses of CNP in the two groups suggest CNP production is dependent on growth plate activity and not a direct effect of TH on CNP gene expression. Our findings suggest novel mechanisms underlying changes in skeletal response during treatment in children with acquired thyroid disease.

Conservation of a growth hormone-responsive promoter element in the human and mouse acid-labile subunit genes.

During extrauterine life, insulin-like growth factors (IGFs) circulate in a ternary serum complex with one IGF-binding protein-3 (IGFBP-3) or IGFBP-5 protein and with a single acid-labile subunit (ALS). GH increases levels of this ternary complex; in mice, this effect is achieved in part by the ability of GH to stimulate mouse ALS (mALS) transcription through an interferon-gamma-activated sequence-like element (GLE) in the mALS promoter. To begin studying how GH regulates human ALS (hALS) gene expression, we cloned the hALS gene and found that it spans approximately 3.3 kb of DNA at chromosomal region 16p13.3. The hALS gene has two exons separated by a 1235-bp intron, which is found at the identical site in rat and mouse ALS genes. Sequence analysis reveals that the hALS 5'-flanking sequence is homologous to the mALS promoter, and that the GH-responsive GLE in the mALS promoter is conserved in both sequence and location in the hALS gene. The region spanning from -755 to -4 bp 5' to the hALS ATG translation start codon directs expression of a luciferase reporter gene in primary rat hepatocytes, and GH increases reporter expression in the presence of the native, but not a mutant, GLE in the hALS promoter. These data suggest that GH stimulates hALS and mALS gene expression by a similar mechanism, which involves at least in part a conserved GLE in the ALS promoter.

Expression of the components of the insulin-like growth factor axis across the growth-plate.

Linear bone growth occurs as the result of proliferation and differentiation of growth-plate chondrocytes. These two phases of chondrocyte growth are regulated separately, with insulin-like growth factor I (IGF-I) being the primary stimulator of proliferation. We studied the expression of the components of the growth hormone GH/IGF system to learn if this proliferative signal is altered as chondrocytes undergo differentiation. Growth-plate chondrocytes were isolated from fetal cows and fractionated on discontinuous Percoll gradients. Five populations were recovered, ranging from high density cells (proliferative chondrocytes) to low density cells (hypertrophic chondrocytes). Messenger RNAs (mRNAs) were analyzed by a reverse transcriptase/quantitative polymerase chain reaction (RT/qPCR) technique. Results showed that mRNA of IGF-I and IGF-II in proliferative chondrocytes was 32 and five fold more abundant, respectively, than in hypertrophic chondrocytes. Of the four major IGF-I mRNA transcripts, the class 1-Ea transcript was predominant. Messenger RNA levels for IGFBP-3, -4, and -5 were also reduced in hypertrophic chondrocytes. Levels of GH receptor, the type 1 IGF receptor, and IGF binding protein-2 (IGFBP-2) mRNAs were unchanged across the growth-plate. Since IGF-I and -II are potent stimulators of proliferation, the down-regulation of these genes may be necessary in order for hypertrophy to proceed.

A single-sample, subcutaneous gonadotropin-releasing hormone test for central precocious puberty.

OBJECTIVE: We compared a rapid, subcutaneous (SQ), single-sample gonadotropin-releasing hormone (GnRH) stimulation test with the standard multiple-sample, intravenous (IV) GnRH stimulation test used in the evaluation of central precocious puberty (CPP). METHODS: We evaluated 22 patients presenting with evidence of precocious puberty. GnRH (100 microg) was administered subcutaneously in the clinic setting with single serum luteinizing hormone (LH) measured 40 minutes after injection. A standard IV GnRH stimulation test was performed within 2 weeks, with serum LH obtained at 0, 20, 40, and 60 minutes. LH was assayed by immunochemiluminometric assay. RESULTS: The mean peak LH levels after IV and SQ testing were identical. A significant correlation (r = .88) was found between the LH determined by SQ stimulations and the peak LH determined by IV GnRH testing. CPP was diagnosed (LH, >/- 8 IU/L) by both SQ and IV testing in 7 of 22 patients and was excluded by both tests in 14 of 22 patients. A diagnostic discrepancy between peak IV and SQ results was seen in 1 patient. CONCLUSIONS: We conclude that mean GnRH-stimulated LH levels from rapid SQ and standard IV testing are indistinguishable and that individual LH levels by each method are strongly correlated. A rapid SQ GnRH test is a valid tool for laboratory confirmation of CPP.

Chondrocytes from osteoarthritic cartilage have increased expression of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and -5, but not IGF-II or IGFBP-4.

Osteoarthritis is a disease in which articular cartilage metabolism is altered, leading to cartilage destruction. As insulin-like growth factor-I (IGF-I) is the major anabolic mediator for articular cartilage, and the IGF-binding proteins (IGFBPs) are an integral part of the IGF axis, they may play a role in the pathophysiology of osteoarthritis. Chondrocytes isolated from fibrillated and normal appearing areas of osteoarthritic human cartilage and from normal cartilage were studied for IGF and IGFBP expression. IGF and IGFBP messenger ribonucleic acids were analyzed by a RT-quantitative PCR technique and Northern blotting. In osteoarthritic chondrocytes, IGF-I message was increased 3.5-fold, IGFBP-3 was increased 24-fold, and IGFBP-5 was increased 16-fold over normal chondrocytes. Chondrocytes from normal appearing areas of cartilage from osteoarthritic joints had intermediate levels. Message levels for beta-actin, IGF-II, and IGFBP-4 were unchanged between the cartilage types. IGF and IGFBP production were analyzed by Western ligand blots and RIAs of conditioned medium from cartilage cultured in serum-free conditions. IGF-I was undetectable in conditioned medium from normal cartilage and increased in that from osteoarthritic cartilage. Osteoarthritic cartilage samples produced IGFBP-2, -3, and -4; glycosylated IGFBP-4; and IGFBP-5. IGFBP-2, -3, and -5 production was increased in osteoarthritic cartilage. Proteases with activity against IGFBP-3 and -5 were also produced by osteoarthritic cartilage. The observation that IGFBP-3 and -5 expression and production are elevated in osteoarthritic cartilage suggests that they may be acting as a competitor for IGF-I in osteoarthritic cartilage, thus reducing the anabolic stimulation of this tissue and contributing to the net loss of cartilage in this disease.

Interleukin-1 and tumor necrosis factor-alpha increase insulin-like growth factor-binding protein-3 (IGFBP-3) production and IGFBP-3 protease activity in human articular chondrocytes.

IGF-I is the major anabolic factor for cartilage matrix production. Chondrocytes and cartilage treated with interleukin-1 alpha (IL-1 alpha), and chondrocytes from several models of inflammatory joint disease, exhibit reduced responsiveness to IGF-I. Since the IGF-binding proteins (IGFBPs) modulate the effects of IGF-I, we examined the effect of IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) on IGFBP production by normal human articular chondrocytes in primary culture. Western ligand blots and immunoprecipitation of conditioned medium samples showed that articular chondrocytes produced IGFBPs-2, -3 and -4 and glycosylated IGFBP-4. Both IL-1 alpha and TNF-alpha increased chondrocyte production of IGFBP-3, but did not alter IGFBP-4 production. The activity of a neutral metalloprotease with the ability to cleave IGFBP-3 was also increased by IL-1 alpha. These data suggest that the cytokines IL-1 alpha and TNF-alpha may act to reduce IGF-I access to chondrocytes by increasing production of IGFBP-3. This may be a factor in the decreased matrix production in the inflammatory arthritides.

A quantitative assay for IGF-I and IGF binding protein mRNAs: expression in malignant melanoma cells.

The quantification of messenger RNA is central in studies of gene expression. We describe a quantitative assay for specific mRNAs (QASM) that measures mRNAs for insulin-like growth factor-I, IGF binding proteins (IGFBPs) -2, -3, -4, and -5, and beta-actin. The assay utilizes reverse transcription and polymerase chain reaction, followed by an ELISA based DNA assay technique. The use of internal (competitive) quantification standards gave poorly linear results, while external standards gave linear and reproducible results. QASM results correlated with IGFBP protein concentrations in conditioned medium and with mRNA levels determined by Northern blotting. QASM was used to study IGFBP expression in human malignant melanoma cells. Messenger RNA for IGFBP-2, -3, and -5 were present, while IGF-I and IGFBP-4 mRNAs were not detected. IGFBP-2 and -3 expression was increased in a dose dependent manner by treatment with IGF-I. Protein concentrations in conditioned media paralleled mRNA levels. QASM is a sensitive, specific, and reproducible approach to determining mRNA levels.

Production and hormonal regulation of insulin-like growth factor binding proteins in bovine chondrocytes.

Linear growth results from proliferation and differentiation of chondrocytes within the growth plates and is regulated, in part, by the insulin-like growth factors (IGFs). IGF binding proteins (IGFBPs) also appear to play a significant, but yet unclear, role. To examine IGFBP production by chondrocytes, we isolated bovine chondrocytes from adult articular, fetal articular, and fetal growth plate cartilage, and maintained them in primary culture as high-density monolayers or encapsulated in alginate beads. Cells were cultured in serum-free conditions with human GH (hGH), insulin, hIGF-I, or hIGF-II. Human IGF-I resulted in higher DNA content in all three of the chondrocyte types. Conditioned medium samples were analyzed for IGFBPs by Western ligand blotting. Chondrocytes released IGFBPs of 24, 29, 33, 39, and 43 kilodaltons (kDa). Deglycosylation and immunoblotting identified the 39/43-kDa doublet as IGFBP-3 and the 33-kDa band as IGFBP-2. All chondrocyte types released 29- and 24-kDa IGFBP bands constitutively. Adult articular chondrocytes increased production all IGFBPs in response to IGF-I, but particularly the 29-kDa band (17-fold increase). Fetal articular chondrocytes showed a similar pattern, but with less of an increase when treated with IGF-I. Fetal growth plate chondrocytes primarily showed increases in IGFBP-3 and the 24-kDa form (4.7- and 2.7-fold, respectively) in response to IGF-I. Although the role of IGFBPs in IGF mediation of articular and growth plate chondrocyte metabolism requires further research, we show here that bovine chondrocytes produce IGFBPs, and the IGFs regulate this production.