Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

doi:10.1016/0167-4889(87)90153-4

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Volume 930, Issue 1, 19 August 1987, Pages 39-47

https://doi.org/10.1016/0167-4889(87)90153-4 Get rights and content

Abstract

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [¹⁴C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [¹⁴C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.

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Gaping of pectoralis minor muscles: Magnitude and characterization of an emerging quality issue in broilers
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Among them, the significantly lower glutamine concentration found in MOD and SEV muscles might be related to an altered and reduced collagen synthesis (Bellon et al., 1995) that could partially explain the phenotype associated to the development of this defect. Indeed, in an in vitro study carried out on human fibroblasts, an increased concentration of glutamine led to a higher collagen synthesis through both a direct stimulatory effect and a concurrent action as a precursor of proline and hydroxyproline (Bellon et al., 1987). In the same time, the significant variations observed in the metabolites involved in energy-generating pathways reinforce the hypothesis that the development of gaping is associated with alterations in post-mortem metabolism.
Recently, a certain number of broiler abattoirs located in different Countries around the World have signaled an emerging quality issue termed “gaping” because of the separation of the fiber bundles affecting the external portion of the bipinnate pectoralis minor muscle. Thus, after defining the criteria to classify the muscles as Normal (NORM), Moderate (MOD), or Severe (SEV) cases, the incidence of gaping under commercial conditions was assessed on a total of 8,600 P. minor obtained from broiler chickens belonging to 43 flocks during a 6-mo period. Then, a total of 180 P. minor were selected based on previously defined criteria to evaluate the main quality traits (pH, color, water-holding/-binding capacity and tenderness), proximate composition, water mobility, and thermal properties as well as metabolic profile through ¹ H-Nuclear Magnetic Resonance spectroscopy. The average incidence of gaping defect was found to be 16.8% (8.8 and 8.0% MOD and SEV cases, respectively). As for the main quality traits, a reduction in ultimate pH was observed as the severity of the gaping defect increased, with SEV muscles displaying significantly lower values in comparison with NORM (5.96 vs. 6.02; P < 0.01), while MOD showed intermediate values (5.99). Concurrently, if compared with their NORM counterpart, MOD and SEV exhibited higher lightness (53.6 and 54.2 vs. 51.8; P < 0.01) coupled with higher (P < 0.05) cooking losses and longer (P < 0.05) transversal relaxation time of extra-myofibrillar water fraction. Overall, no significant differences were found concerning proximate composition and thermal properties. With regard to the metabolic profile, a significantly lower (P < 0.001) glutamine concentration was found in MOD and SEV muscles that, concurrently, revealed significant (P < 0.05) variations in the metabolites involved in energy-generating pathways. Overall, these findings evidenced that the gaping defect affecting broilers’ P. minor muscles have strong similarities with the pale-soft-exudative condition previously described in poultry and likely results from the biochemical processes taking place during the post-mortem conversion of muscle to meat.
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Collagen production is another key function of activated myofibroblasts and the ability of ITE and FICZ to block TGF-β–induced collagen production was tested by slot blot (Figure 4B). To promote collagen production in orbital fibroblasts, cultures were supplemented with l-proline and ascorbic acid because functional collagen requires these key biosynthetic precursors.27,28 Collagen levels in culture supernatants were induced approximately fourfold by TGF-β, and both ITE and FICZ significantly attenuated collagen induction.
Thyroid eye disease (TED) is a degenerative disease that manifests with detrimental tissue remodeling, myofibroblast accumulation, and scarring in the orbit of affected individuals. Currently, there are no effective therapies for TED that target or prevent the excessive tissue remodeling caused by myofibroblast formation and activation. The canonical cytokine that induces myofibroblast formation is transforming growth factor (TGF)-β. The TGF-β signaling pathway is influenced by aryl hydrocarbon receptor (AHR) signaling pathways. We hypothesized that AHR agonists can prevent myofibroblast formation in fibroblasts from patients with TED, and thus AHR ligands are potential therapeutics for the disease. Orbital fibroblasts explanted from patients with TED were treated with TGF-β to induce myofibroblast formation, contraction, and proliferation. We found that AHR ligands prevent TGF-β–dependent myofibroblast formation, and this ability is dependent on AHR expression. The AHR and AHR ligands block profibrotic Wnt signaling by inhibiting the phosphorylation of GSK3β to prevent myofibroblast formation. These results provide new insight into the molecular pathways underlying orbital scarring in TED. These novel studies highlight the potential of the AHR and AHR ligands as future therapeutic options for eye diseases and possibly also for other scarring conditions.
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Transforming growth factor-β₁ (TGF-β₁) plays a central role in promoting extracellular matrix protein deposition by promoting the transformation of fibroblasts to myofibroblasts. To gain new insights into the transcriptional programs involved, we profiled human fetal lung fibroblast global gene expression in response to TGF-β₁ up to 24 hours using oligonucleotide microarrays. In this report, we present data for 146 genes that were up-regulated at least twofold at two time points. These genes group into several major functional categories, including genes involved in cytoskeletal reorganization (n = 30), matrix formation (n = 25), metabolism and protein biosynthesis (n = 27), cell signaling (n = 21), proliferation and survival (n = 13), gene transcription (n = 9), and of uncertain function (n = 21). For 80 of these genes, this is the first report that they are TGF-β₁-responsive. The early induction of two members of the inhibitor of differentiation (ID) family of transcriptional regulators, ID1 and ID3, was followed by the up-regulation of a number of genes that are usually expressed by highly differentiated smooth muscle cells, including smooth muscle myosin heavy chain, basic calponin, and smoothelin. These findings were confirmed at the protein level for primary adult lung fibroblasts. ID1 further behaved like a typical immediate-early gene and, unlike ID3, was expressed and induced at the protein level. Immunohistochemical analysis showed that ID1 was highly expressed by (myo)fibroblasts within fibrotic foci in experimentally induced pulmonary fibrosis. ID1 acts as a dominant-negative antagonist of basic helix-loop-helix transcription factors that drive cell lineage commitment and differentiation. These findings have important implications for our understanding of fibroblast transcriptional programming in response to TGF-β₁ during development, oncogenesis, tissue repair, and fibrosis.

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Regular paperEffects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

Abstract

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Collagen Rel. Res.

J. Biol. Chem.

Metab. Clin. Exp.

J. Biol. Chem.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

J. Chromatog.

Anal. Biochem.

Anal. Biochem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Regular paper
Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures