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Visual demonstration of growth hormone receptors on human growth plate chondrocytes
G A Werther1, K M Haynes, R Barnard
1Department of Endocrinology and Diabetes, Royal Children's Hospital, Melbourne, Victoria, Australia.
Insights
Growth hormone (GH) receptors are present in human infant growth plates, including bone and blood cell precursors. Receptor expression is linked to maturation and declines after puberty.
Area of Science:
- Endocrinology
- Developmental Biology
- Histology
Background:
- The precise locations where growth hormone (GH) acts in human infants are not fully understood.
- Animal studies suggest direct effects of GH on growth plates and other tissues.
Purpose of the Study:
- To identify and localize GH receptors in the human infant growth plate using a specific monoclonal antibody.
- To investigate the presence and distribution of GH receptors in various cell types within the infant growth plate.
Main Methods:
- Immunocytochemistry was used to stain sternochondral cartilage sections and cultured chondrocytes from infants with a monoclonal antibody against the human GH receptor (MAb 263).
- Radioligand binding assays with [125I]human GH ([125I]hGH) and emulsion autoradiography were performed on cultured chondrocytes.
- Cartilage from adolescents was also analyzed for comparison.
Main Results:
- GH receptors were detected in chondrocytes (proliferative and hypertrophic layers), perichondrium, osteocytes, and hemopoietic precursor cells in infant growth plate sections.
- Cultured infant chondrocytes showed heterogeneous GH receptor staining that changed over time.
- [125I]hGH binding was confirmed in infant chondrocyte cultures, with MAb 263 showing 35% displacement of [125I]hGH.
- Adolescent cultured chondrocytes did not exhibit specific [125I]hGH binding.
Conclusions:
- GH receptors are widely distributed in mesenchymal cells of the human infant growth plate, including bone and hematopoietic precursors.
- GH receptor expression is dependent on maturation in both intact tissue and cell culture.
- Receptor expression appears to decrease after the pubertal growth phase.
Abstract:
The sites of action of GH in the human infant remain unclear; recent evidence in animals suggests direct actions on growth plate and other tissues. We have used a monoclonal antibody recognizing the human GH receptor to visually identify and localize GH receptors in the human infant growth plate. Sternochondral cartilage was obtained at postmortem from infants dying of sudden infant death (n = 20), and either decalcified, fixed, and cut into longitudinal sections or digested with collagenase for monolayer culture of chondrocytes. Sections of cultured chondrocytes were stained immunocytochemically with a monoclonal antibody recognizing human GH receptor (MAb 263), using an avidin-biotin system. Sternochondral cartilage was also obtained at operation from adolescents undergoing sternochondroplasty. In infant tissue, GH receptor was identified in sections in chondrocytes of the proliferative and hypertrophic layers, in perichondrium, in osteocytes in new bone, and in hemopoietic precursor cells in marrow. Cultured chondrocytes showed heterogeneous staining for GH receptor. With prolonged culture from 5-8 days, the pattern of staining changed from individual cells to groups of cells. [125I]Human (h)GH showed specific binding to chondrocyte monolayer (0.6 +/- 0.3%), confirmed visually on emulsion autoradiography. In support of specificity of MAb263, it was able to displace [125I]hGH from monolayers by 35%. Adolescent cultured chondrocytes failed to demonstrate specific binding of [125I]hGH. We conclude that GH receptors are widely distributed in a range of mesenchyme cells in the human infant growth plate, including bone and hemopoietic precursors. The expression of these receptors appears to be maturation dependent in both intact tissue and culture, while they may no longer be expressed after the peak growth phase of puberty.
