The Clinical Laboratory Evaluation of GH Responsiveness

Philip A. Gruppuso

Department of Pediatrics, Rhode Island Hospital and Brown University Medical School, Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Philip A. Gruppuso, M.D., Department of Pediatrics, 593 Eddy Street, Rhode Island Hospital, Providence, Rhode Island 02903.

The recent history of pediatric endocrinology is dominated by an effort to develop diagnostic studies that can identify patients with pathological short stature among the large population of normal, short children. With the availability of recombinant human GH came the opportunity to treat children who have short stature of unclear etiology. Defining the cause of short stature became important in determining the appropriateness of GH therapy and the expectations for a salutary effect on final height.

The physiological system involving GH, the IGFs, their receptors, and the IGF-binding proteins (IGFBPs) has been the subject of intensive basic and clinical investigation during the last several decades (1). The central role of IGF-I in mediating many of the effects of GH is illustrated by the consistent phenotypic features exhibited by abnormalities throughout this system. The complexity of the GH/IGF axis with its many components provides for numerous, distinct pathophysiological mechanisms that are still being defined. Primary pituitary abnormalities are relatively amenable to direct diagnosis, with a combination of provocative GH testing and neuro-imaging often providing a clear indication of GH deficiency. Neuorendocrine abnormalities associated with impaired GH secretion are perhaps the most frequently diagnosed endocrine causes of short stature. When subtle and unassociated with global pituitary dysfunction, these disorders are difficult to diagnose.

Like GH deficiency, abnormalities in GH responsiveness manifest as IGF-I deficiency. These disorders have also come under close scrutiny in recent years. Laron et al. (2) suggested that these disorders be classified as primary or secondary. Perhaps the best characterized secondary cause of impaired GH responsiveness is the attenuation of IGF-I production seen in nutritional deprivation (3). Other causes include renal disease, liver disease, and the presence of antibodies to GH or the GH receptor. Primary defects in GH responsiveness occur much less frequently than secondary defects. The prototype for these disorders, first described by Laron et al. (4), is GH resistance associated with inactivating mutations of the GH receptor. However, abnormalities of GH signaling at any point along the GH signaling pathway can result in the GH-deficient phenotype through attenuation of IGF-I production. Thus, there has been considerable interest in abnormalities in GH signal transduction as a cause of idiopathic short stature.

Considerable progress has been made in characterizing the mechanism for GH signaling (5). The GH receptor encodes a transmembrane protein with a cytoplasmic domain that does not itself encode for a signaling mechanism. However, the binding of GH to its receptor promotes the tyrosine phosphorylation of the receptor as well as other cellular proteins. Furthermore, tyrosine kinase activity can be recovered in a highly purified GH receptor preparation. These discordant observations were reconciled by the discovery that the nonreceptor tyrosine kinase, JAK2, associates with the GH receptor on GH binding, and that this association leads to JAK2 activation.

GH receptor-mediated JAK2 activation seems to be involved in the spectrum of GH actions. The STAT (signal transducers and activators of transcription) family of transcription factors seems to play a key role in mediating GH effects. JAK2-mediated phosphorylation of several members of this family—including STATs 1, 3, 5A, and 5B—leads to their dimerization and nuclear translocation. Once in the nucleus, activated STATs appear to be involved in many of the gene regulation events associated with GH action. However, STAT activation alone is not sufficient to explain the physiological actions of GH. Current research indicates that multiple signaling pathways and transcription factors act downstream of the GH receptor. These pathways are probably required for not only the well-characterized induction of IGF-I and many of the IGFBPs, but also for the insulin-like and counter-regulatory metabolic effects of GH.

A key downstream event in GH-mediated growth stimulation is the transcriptional activation of IGF-I. Unfortunately, the transcriptional regulation of the IGF-I gene is extraordinarily complex and has resisted elucidation. Although abnormalities in the IGF-I promoter causing its dysregulation have not been identified as a cause of IGF-I deficiency, there is every reason to theorize that such patients exist. A recent report (6) demonstrating that mutations in the CTNS promoter can account for cystinosis in a subset of patients without coding region mutations may prove to be a prototype for other disorders, including IGF-I deficiency. Another consequence of the complexity of IGF-I promoter function is that it may limit the usefulness of IGF-I induction as a readout for GH action. For example, transcriptional activation of the IGF-I gene is differentially affected by estrogen, which stimulates IGF-I expression in the uterus but inhibits GH-mediated induction in the liver (7).

When considering the evaluation of GH nonresponsiveness, at least two other factors deserve comment. The first is that the clinical laboratory evaluation of GH action does not assess GH effect at the relevant target tissues. Considerable evidence supports the model that GH induces the local production of IGF-I that acts as a paracrine and autocrine factor in critical target tissues, including bone and muscle (1). Thus, the measurement of circulating IGF-I, which is hepatic in origin, is a surrogate for evaluating target tissue responsiveness. Secondly, GH signaling is subject to rapid and potent modulation in response to physiological perturbations. A recently identified mechanism for attenuation of GH signaling involves members of a family of cytokine-inducible genes identified by the acronym SOCS (suppressors of cytokine signaling). Available evidence indicates that the several SOCS family members induced by GH can inhibit JAK- mediated GH signaling, thus providing for a negative feedback loop (5). In addition, pathophysiological stress, such as that associated with infection or inflammation, may lead to GH resistance through cytokine-mediated induction of SOCS family members (8).

Given the complexity of GH signaling and the potential for physiological and pathophysiological modulation, one might expect that the clinical laboratory assessment of GH responsiveness would be challenging. The GH dependence of IGF-I and IGFBP-3 has been widely applied to the evaluation of children with subnormal growth velocity. Age- and pubertal stage-dependent norms for circulating IGF-I and IGFBP-3 levels have been widely applied to the initial evaluation of these children (9). However, such measurements cannot distinguish between patients with GH deficiency and impaired GH responsiveness. Therefore, tests for GH receptor abundance, activity, and physiological function would be useful in the evaluation of patients with the IGF-I deficiency phenotype who do not appear to have GH deficiency.

The primary serum GH-binding protein (GHBP) in humans is derived by proteolytic cleavage from the extracellular domain of the GH receptor. Therefore, it is not surprising that GHBP was found to be absent from the circulation of patients with GH receptor deficiency (10, 11). Measurement of serum GHBP has been examined in patients with idiopathic short stature (12) and has been proposed as a possible indicator of partial GH insensitivity. However, this test does not have established clinical use for this purpose. GHBP levels may be subject to ligand-mediated down- regulation of the GH receptor. Furthermore, an indicator of receptor abundance would not be useful for identifying patients with downstream abnormalities in the GH/IGF-I axis. However, all of these issues would be addressed by a reproducible provocative test of GH response.

Over the last 20 yr, many investigators have examined the clinical use of a variety of IGF-I and IGFBP-3 generation tests to evaluate GH responsiveness. In a recent issue of JCEM, Buckway et al. (13) studied the IGF-I generation test as an indicator of GH sensitivity. In addition to studying a large population of normal and GH-deficient subjects, they included subjects derived from a well-defined Ecuadorian population that has a splice mutation affecting the extracellular domain of the GH receptor. This ambitious and carefully carried out study showed that a standardized IGF-I generation test had relatively low sensitivity (77%) in detecting impaired GH responsiveness in patients with known defects in the GH receptor. Furthermore, GH response in GH-deficient subjects overlapped with that of patients who had documented receptor defects. Such a result may not bode well for the usefulness of the IGF-I generation test in detecting subtle abnormalities in GH responsiveness.

In this issue of JCEM, Jorge et al. (14) report data on the reproducibility of IGF-I and IGFBP-3 generation tests. They studied 12 children with idiopathic short stature and a prior demonstration of GH sufficiency, based on provocative testing. The authors performed molecular analyses of the GH receptor gene to eliminate patients with known inactivating mutations in the GH receptor. They performed IGF-I and IGFBP-3 generation tests, using a standardized procedure proposed by Blum et al. (15). After observing discordance in the results of several repeat generation tests, they went on to perform repeat testing after intervals ranging from 1–22 months on all of the patients. Test results, whether assessed as IGF-I or IGFBP-3 induction in response to GH, were discordant between the two tests about half the time. Although the authors found some differences between IGF-I and IGFBP-3 responses, the clearest conclusion from their studies is that the IGF-I and IGFPB-3 generation tests are of questionable value in diagnosing partial GH insensitivity in a population of patients without GH receptor defects.

This conclusion may not be very surprising to pediatric endocrinologists who have followed the history of clinical testing of the GH/IGF axis. Provocative tests for GH deficiency, even with the application of recently developed technology (16), are still a source of controversy because of variation in assays, limited discretion in their application, and a lack of standardization of results. Assessment of normal, endogenous GH secretion through overnight or 24-h GH profiles has not proven to be a definitive test for GH deficiency for similar reasons as well as questionable reproducibility and specificity (9). Static measurements of IGF-I and IGFBP-3 are clearly useful as screening tests but are not considered to be definitive tests, in part due to the factors other than GH sufficiency that contribute to their circulating levels. Similarly, circulating GHBP levels are limited by the multifactorial regulation of GH receptor cell surface abundance and the prospect of defects in postreceptor signaling. It now appears that tests of GH responsiveness may have limitations in identifying patients with intermediate degrees of GH insensitivity. Both a lack of specificity and reproducibility may limit the usefulness of these tests. Thus, pediatric endocrinologists will have to continue to apply their clinical judgment to the evaluation of short children without an immediate prospect for a definitive clinical laboratory test of GH signaling.

Footnotes

Abbreviations: GHBP, GH-binding protein; IGFBP, IGF-binding protein; SOCS, suppressors of cytokine signaling; STAT, signal transducers and activators of transcription.

Received November 20, 2001.

Accepted November 20, 2001.

References

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