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Quantification of rib COL1A2 gene expression in healthy and fluorosed Inner Mongolia cashmere goats.
| Fluoride | Li W, Yang L, Ren Y, Yan X, Wang J. | 40(1):13-18.
China Collagen Inner Mongolia Ribs Bones/Joints Mechanism of Toxicity Skeletal Fluorosis Animal Study
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Abstract

Damage to collagen protein and its gene expression caused by excessive fluoride (F) ingestion plays an important role in the etiology of skeletal fluorosis. Recently we found that industrial F pollution significantly increased the expression level of type II collagen gene (COL2A1) in rib cartilage of Inner Mongolia cashmere goats. With the same goats and methods, we have now quantified another important collagen gene, the rib COL1A2 gene, which encodes an a2(I) polypeptide chain assembled into collagen molecules. The results showed that the expression level of COL1A2 and COL1A2/B-actin increased by 88% and 81%, respectively.

EXCERPTS:

INTRODUCTION

Skeletal fluorosis is a well-known chronic metabolic bone disease caused by ingesting excessive amounts of fluoride (F), especially from water, and usually or less often from food and air. The unique mechanical properties of bone depend on the composition and organization of its extracellular matrix, and collagens are major structural components of the extracellular matrix.1 As suggested by Susheela in the 1970s, collagen protein is a target damaged by excessive F ingestion.2 Thus, investigation of the effect of F on collagen protein by quantification of some related collagen gene expressions should help us understand the molecular mechanism of F toxicity.

A number of reports show that F affects the collagen metabolism of cartilage and bone.3-6 In our previous work, we also found that the collagen of fluorosed tooth matrix had undergone obvious morphological changes and the total amino acid content of the fluorosed matrix was significantly greater than that of nonfluorosed control tooth matrix.7-11 Furthermore, we recently reported that industrial fluoride pollution increases the expression level of type II collagen gene (COL2A1) that encodes the cartilage-specific collagen in rib cartilage.12

In ribs the predominant form of collagen is collagen type I encoded by COL1A2 gene. The question then arises: Can F also increase the COL1A2 gene expression in ribs? Using the same goats studied in our recent report,12 we developed and validated real-time RT-PCR (reverse transcription polymerase chain reaction) assays13 to quantify the expression level of COL1A2 gene in the ribs of Inner Mongolia cashmere goats.

MATERIALS AND METHODS

The Inner Mongolia cashmere goats and their treatment were the same as described previously.12 The rib tissue preparation and the total RNA extraction were performed following the methods described in that report.12

Two pairs of specific primers (Table 1) were designed according to the alignments of the published cDNA sequences of COL1A2 and ß-actin genes in humans and sheep.

The conventional RT-PCR amplification and identification of the above two gene fragments were also the same as those described previously.12 Real-time RTPCR conditions and analysis of the COL1A2 gene expression level were the same as described previously as well as the statistical analysis.12

DISCUSSION

Although many experimental studies have shown that F can cause structural changes in collagen fibers, little is known about the molecular mechanism of these effects.15-17 It is generally accepted that type I collagen is the main type of collagen in the rib responsible for its stability and cell biological functions.18 The type I collagen gene (COL1A2) encodes an a2(I) polypeptide chain that is assembled into a collagen molecule.

In the present study, we quantified the COL1A2 gene expression level in ribs of Inner Mongolia cashmere goats by real-time RT-PCR. The results showed an 88% increase in COL1A2 expression and an 81% increase in COL1A2/B-actin expression.

Our findings thus confirm that industrial fluoride pollution increases not only the expression level of COL2A1 gene but also that of the COL1A2 gene in Inner Mongolia cashmere goats. Accordingly, we suggest that enhanced expression levels of the COL2A1 and COL1A2 gene are involved in skeletal fluorosis. Thus, it appears that F interferes with the balance between the synthesis and degradation of normal collagen by inducing the synthesis of large quantities of imperfect collagen or noncollagenous protein.

Finally, we note that the regulation of gene expression is complex and involves both transcriptional and post-transcriptional mechanisms.19 Our initial results with the COL2A1 and COL1A2 gene expression remain to be verified with further studies. For this purpose, our research group is carrying out experimental studies of collagen protein expression in rabbits and rats.

ACKNOWLEDGEMENT

This research was sponsored by the China National Natural Science Foundation (Grant No. 30471303).

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