Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells

The effect of hypoxia on the proliferation and collagen synthesis of cultured rat mesangial cells was examined under normal-glucose (NG, 5 mM) and high-glucose (HG, 25 mM)-media conditions. In addition, a role for osteopontin (OPN) in mediating these processes was assessed. Quiescent cultures were exposed to hypoxia (3% O2) and normoxia (18% O2) in a serum-free medium with NG or HG, and cell proliferation, collagen synthesis, and OPN expression were assessed. Cells exposed to hypoxia in NG medium resulted in significant increases in [3H]thymidine incorporation, cell number, and [3H]proline incorporation, respectively. HG incubations also produced significant stimulation of these parameters under normoxic conditions, which were markedly enhanced in cells exposed to hypoxia in HG medium. In addition, hypoxia and HG stimulated the mRNA levels of type IV collagen, and the combination of hypoxia and HG resulted in additive increases in type IV collagen expression. Hypoxia and HG also stimulated OPN mRNA and protein levels in an additive fashion. A neutralizing antibody to OPN or its β3-integrin receptor significantly blocked the effect of hypoxia and HG on proliferation and collagen synthesis. In conclusion, these results demonstrate for the first time that hypoxia in HG medium produces exaggerated mesangial cell growth and type IV collagen synthesis. In addition, OPN appears to play a role in mediating the accelerated mesangial cell growth and collagen synthesis found in a hyperglycemic and hypoxic environment.

Download Full-text

Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells

Biochemical Journal ◽

10.1042/bj2450235 ◽

1987 ◽

Vol 245 (1) ◽

pp. 235-241 ◽

Cited By ~ 18

Author(s):

A Oikarinen ◽

T Salo ◽

L Ala-Kokko ◽

K Tryggvason

Keyword(s):

Total Protein ◽

Messenger Rna ◽

Collagen Synthesis ◽

Glucocorticoid Receptors ◽

Type Iv Collagen ◽

Cell Layer ◽

Specific Binding ◽

Specific Radioactivity ◽

Filtration Method ◽

Type Iv

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.

Download Full-text

The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction.

Journal of Experimental Medicine ◽

10.1084/jem.176.6.1571 ◽

1992 ◽

Vol 176 (6) ◽

pp. 1571-1576 ◽

Cited By ~ 49

Author(s):

E P Peten ◽

L J Striker ◽

M A Carome ◽

S J Elliott ◽

C W Yang ◽

...

Keyword(s):

Gene Expression ◽

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Collagen Synthesis ◽

Type Iv Collagen ◽

Chain Reaction ◽

Collagen Mrna ◽

Type Iv ◽

Polymerase Chain

We previously reported that one of the main components of the sclerotic material in human glomerular diseases was type IV collagen. In this study we examined the contribution of increased synthesis to this process at the gene expression level. Sufficient material has not been available to study type IV collagen synthesis by normal or sclerotic glomeruli in humans. We took advantage of the availability of nephrectomy specimens from patients with renal carcinoma, and of the observation that approximately 50% of these patients develop varying degrees of glomerulosclerosis. We microdissected glomeruli from 10 patients and analyzed them using in situ reverse transcription coupled with polymerase chain reaction (PCR) analyses (in situ RT-PCR). alpha 2IV collagen mRNA, after reverse transcription into cDNA, was detected in all patients and appeared to be increased in those with glomerulosclerosis (n = 5). A competitive PCR assay was developed to quantitate this change. There was an average 3.7-fold increase in glomerular type IV collagen cDNA in patients with significant sclerosis. This change was not due to an increased number of glomerular cells. Thus, glomerulosclerosis in humans is associated with an elevation of glomerular type IV collagen gene expression, suggesting that increased synthesis of type IV collagen may represent one component of this process.

Download Full-text

Selective estrogen receptor modulators suppress mesangial cell collagen synthesis

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.2.f309 ◽

2000 ◽

Vol 279 (2) ◽

pp. F309-F318 ◽

Cited By ~ 58

Author(s):

Joel Neugarten ◽

Anjali Acharya ◽

Jun Lei ◽

Sharon Silbiger

Keyword(s):

Estrogen Receptor ◽

Collagen Synthesis ◽

Type Iv Collagen ◽

Mesangial Cell ◽

Type I ◽

Dependent Manner ◽

Type Iv ◽

Estrogen Receptor Modulators ◽

Receptor Modulators ◽

Dose Dependent

Estrogen receptor modulators (SERMs) are “designer drugs” that exert estrogen-like actions in some cells but not in others. We examined the effects of the SERMs LY-117018 (an analog of raloxifene) and tamoxifen on mesangial cells synthesis of type I and type IV collagen. We found that LY-117018 and tamoxifen suppressed mesangial cell type IV collagen gene transcription and type IV collagen protein synthesis in a dose-dependent manner, with a potency identical to that of estradiol. Type I collagen synthesis was also suppressed by LY-117018 in a dose-dependent manner with a potency identical to that of estradiol but greater than that of tamoxifen. Genistein, which selectively binds to estrogen receptor-β in nanomolar concentrations, suppressed type I and type IV collagen synthesis, suggesting that estrogen receptor-β mediates the effects of estrogen on collagen synthesis. Because matrix accumulation is central to the development of glomerulosclerosis, second-generation SERMs may prove clinically useful in ameliorating progressive renal disease without the adverse effects of estrogen on reproductive tissues.

Download Full-text