Mitogenic effects of growth hormone in cultured human fibroblasts. Evidence for action via local insulin-like growth factor I production

We examined human growth hormone's (hGH) effect on mitogenesis in cultured human fibroblasts, and the role of local insulin-like growth factor I (IGF-I). With 0.5% human hypopituitary serum (HPS), hGH increased thymidine incorporation (TI) over serum-free medium dose responsively, with half-maximal effect at 10 ng/ml (0.5 nM) (hGH 127 ± 8.8%; IGF-I 107 ± 1.7% [SEM]) (n = 10). Similarly, with 0.5% HPS, hGH and IGF-I increased cell replication by 172 ± 8.2% and 169 ± 25%, respectively (n = 4). Specific IGF-I monoclonal antibody (Sm1.2) dose dependently blunted TI stimulated by 10 ng/ml hGH or IGF-I (at 1:1000, 38 ± 6.5% and 30 ± 14% reduction, respectively). Sm1.2 also reduced cell replication by both 10 ng/ml hGH and IGF-I, respectively, to 32% and 42% of stimulated values. Dexamethasone (0.1 μM) synergistically enhanced TI by both IGF-I and hGH. A 28-h time course for TI showed that hGH stimulated a similar peak to IGF-I, lagging in its effect by 4-10 h. We have provided further evidence that hGH stimulates growth of cultured human fibroblasts via local IGF-I production, consistent with IGF-I's paracrine-autocrine role.J. J. Cook, K. M. Haynes, and G. A. Werthe

Visual demonstration of growth hormone receptors on human growth plate chondrocytes

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

Demonstration and localization of growth hormone receptor in human skin and skin fibroblasts

Clinical evidence suggests that skin is responsive to GH status in vivo. We sought to demonstrate the presence of GH receptors in human skin and in cultured skin fibroblasts using the techniques of immunohistochemistry and northern blotting

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.<br /

Immunoreactive growth hormone receptor/binding protein is present on fibroblasts and in serum of Laron-type dwarfs

Laron-type dwarfism is an autosomal recessive disorder characterised by extreme growth retardation and growth hormone (GH) resistance and has been shown in some cases to be associated with mutations in the GH receptor gene. Limited data suggest that in this condition specific liver GH binding is absent. In the majority of reported cases specific GH binding is also absent in serum. However it is not known whether the GH receptor and/or the serum GH binding protein are expressed in this condition