A critical concept in understanding growth hormone activity is that it has two distinct types of effects:. Keeping this distinction in mind, we can discuss two major roles of growth hormone and its minion IGF-I in physiology.
Growth is a very complex process, and requires the coordinated action of several hormones. The major role of growth hormone in stimulating body growth is to stimulate the liver and other tissues to secrete IGF-I. IGF-I stimulates proliferation of chondrocytes cartilage cells , resulting in bone growth.
Growth hormone does seem to have a direct effect on bone growth in stimulating differentiation of chondrocytes. IGF-I also appears to be the key player in muscle growth.
It stimulates both the differentiation and proliferation of myoblasts. It also stimulates amino acid uptake and protein synthesis in muscle and other tissues.
Growth hormone has important effects on protein, lipid and carbohydrate metabolism. In some cases, a direct effect of growth hormone has been clearly demonstrated, in others, IGF-I is thought to be the critical mediator, and some cases it appears that both direct and indirect effects are at play. Production of growth hormone is modulated by many factors, including stress, exercise, nutrition, sleep and growth hormone itself. The transcription start site was defined by ribonuclease protection assay.
Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from bp. A minimal bp promoter allowed pituitary-specific expression. You and Your Hormones. Students Teachers Patients Browse. Human body. Home Hormones Growth hormone. Growth hormone Growth hormone is produced by the pituitary gland.
It has many functions including maintaining normal body structure and metabolism. How is growth hormone controlled? What happens if I have too much growth hormone? What happens if I have too little growth hormone? Last reviewed: May Prev.
Gonadotrophin-releasing hormone. Growth hormone-releasing hormone. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant negative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels.
The healthy parents of these children are hemizygous for GH-1 , showing that the presence of a single GH-1 allele is sufficient for GH production and secretion, resulting in normal GH levels in serum 3. In a few patients, splice site mutations in intron IV of GH-1 were described, resulting in skipping the nucleotides coding for amino acids aa — and subsequently in a frame shift in exon 5 4. The mutant GH protein is thus subject to serious structural changes.
In patients harboring this kind of mutation, GH concentrations are extremely low even though one intact GH-1 allele is present. Therefore, the presence of del32—71GH in the somatotrophs causes a blockade of wild-type wt GH secretion by an as yet unknown mechanism. Schematic representation of the wtGH protein tertiary structure. The two intramolecular disulfide bridges and distinct aa positions corresponding to end points of deleted aa stretches are outlined.
When human wtGH and del32—71GH were coexpressed in different cell types, the dominant negative effect was observed only in neuroendocrine cells, whereas no impairment of wtGH secretion was found in other cell types e. Neuroendocrine cells differ from these cells by having specific modes of protein transport, sorting, storage, and release In transient transfection studies involving rat GH 4 C 1 neuroendocrine cells, it was observed that del32—71GH suppresses both intracellular accumulation and secretion of wtGH without itself being accumulated or secreted, whereas wtGH and del32—71GH mRNAs were shown to be present in approximately equal quantities Studies with COS cells revealed an uneven distribution of wtGH and del32—71GH, the former being localized in the Golgi apparatus and the latter retained in the endoplasmic reticulum.
It was suggested that the presence of the misfolded protein causes Golgi apparatus fragmentation, thus disrupting transport from the endoplasmic reticulum to the Golgi apparatus also involving other membrane and secretory proteins These findings in nonneuroendocrine cells were not observed in GH 4 C 1 neuroendocrine cells, showing no deleterious effect of human del32—71GH on the production and secretion of heterologously expressed human prolactin, suggesting that the effects of mutant GH are cell type specific In transgenic mice expressing human del32—71GH, a marked decrease in GH levels was found in pituitary extracts, and the affected animals developed short stature Moreover, multiple anterior pituitary deficiencies, pituitary hypoplasia, morphological abnormalities of somatotrophs with few secretory vesicles, and macrophage hypophyseal invasion were detected.
In contrast, the pituitary abnormalities observed in patients with IGHD II are not as severe as those evident in the in vivo mouse model, because a small to normal size pituitary gland was confirmed by magnetic resonance tomographic analysis Several hypotheses have been discussed in the literature to explain the basic mechanisms of the interference of some mutant GH forms with wtGH. The aim of this in vitro study using GH 4 C 1 cells was to elucidate the importance of specific aa or stretches of aa in the context of the GH tertiary structure for exhibition of the dominant negative effect.
Shorter or longer incubation periods 18, 24, and 72 h, respectively were demonstrated to be inappropriate for the RIA used, because the GH values measured were beyond the detection capacity of the system. The primers were designed to yield products no longer than bp. For each sample, duplicate measurements were performed, and the arithmetic mean was calculated from each duplicate measurement. Threshold cycle values were determined using Bio-Rad iCycler software version 3.
In addition, the image was captured on Kodak film Eastman Kodak Co. Vital and nonvital cells were distinguished under the microscope via staining with 0. Differences between the transfection groups were analyzed by t test.
To investigate the structure-function relationship of hGH mutants with respect to a negative effect on wtGH secretion, a series of hGH constructs that were mutated or deleted in specific aa or stretches of aa was generated. Therefore, diverse deletion mutants, del32—46, del32—52, del32—53, del32—63, and del32—69 were constructed in which the respective aa stretch was deleted.
Of additional interest was the role of the disulfide bridge constituted between Cys 53 and Cys , which is disrupted in del32—71GH. Forty-eight hours posttransfection, GH was measured in incubation media and cell extracts. The degree of GH reduction was proportional to the increase in size of the deletion. The same relationship between the extent of deletion and the amount of detectable hGH was found in protein extracts of the respectively transfected cells Fig. The GH content of each sample was measured twice, using the mean value for calculations.
The extent of deletion played the major role in expression of the dominant negative effect on wtGH. To determine whether the amount of hGH was underestimated in the RIA due to the intrinsic competition with recombinant hGH, cell extracts and media were analyzed using Western blots Fig. Densitometric evaluation of protein bands corresponding to wtGH and mutant GH detected by polyclonal antiserum as well as by monoclonal antibodies directed toward the N terminus of hGH revealed comparable amounts as determined using RIA.
The finding that mutant protein amounts were not underestimated in the RIA was strengthened by the comparability of RIA and Western blot results for cells monotransfected with pwtGH, pdel32—46, pdel32—53, or pdel32—71 data not shown. Protein bands corresponding to the wt and some mutant hGH forms are depicted by arrows and the respective molecular weights. To investigate whether del32—53GH exerts a dominant negative effect as suggested by the above RIA data, we performed parallel cotransfection experiments in which pdel32—53 or empty vector was cotransfected in varying amounts together with pwtGH Fig.
The total amount of transfected plasmid was kept constant by adapting the amount of pwtGH cotransfected. The concentration of total GH was constantly lower for cells cotransfected with pdel32—53GH and pwtGH than for cells cotransfected with empty vector and pwtGH, indicating a dominant negative effect of del32—53GH on wtGH.
This effect became significantly stronger with increasing amounts of pdel32—53GH. The ratio of the respective plasmids used for transfection experiments is depicted on the left. The data reveal a dose-dependent dominant negative effect of del32—53GH on wtGH. The total cell numbers and viability of cells expressing only wtGH or coexpressing wtGH and del32—71GH stayed equivalent in individual experiments over periods of up to 80 h, thus making an acute toxic effect of del32—71GH on the cells unlikely data not shown.
0コメント