Molecular structure of tail tendon fibers in TIEG1 knockout mice using synchrotron diffraction technology

Abstract

The purpose of this study was to characterize the effect of TIEG1 on the molecular structure of collagen within tail tendon fibers using 3-mo-old female C57BL/6 wild-type (WT) and TIEG1 KO mice. Synchrotron X-ray microdiffraction experiments were carried out on single tendon fibers extracted from the WT and TIEG1 KO dorsal tail tendon. The fibers were scanned in the radial direction, and X-ray patterns were obtained. From these patterns, the meridional direction was analyzed through X-ray intensity profile. In addition, collagen content was investigated using hydroxyproline assays, and qualitative real-time PCR experiments were performed on RNA isolated from fibroblasts to examine specific gene expression changes. The results showed different X-ray diffraction patterns between WT and TIEG1 KO tendon fibers, indicating a disorganization of the collagen structure for the TIEG1 KO compared with WT mice. Furthermore, the analyses of the X-ray intensity profiles exhibited a higher (23 A) period of collagen for the TIEG1 KO compared with the WT mice. The results of the hydroxyproline assays revealed a significant decrease in the TIEG1 KO compared with WT mice, leading to a decrease in the total amount of collagen present within the TIEG1 KO tendons. Moreover, qualitative real-time PCR results showed differences in the expression profiles of specific genes known to play important roles in tendon fiber development. These data further elucidate the role of TIEG1 on tendon structure and could explain the previous defects in the structure-function relationship found for TIEG1 KO tendon fibers.

Fig. 1.

Tendon fiber X-ray pattern for wild-type (WT; A) and TIEG1 knockout (KO) (B) mice. C: profiles drawn along the meridional and lateral directions. D: order of the peak represented on the meridional X-ray intensity profile.

Fig. 2.

Meridional X-ray intensity (I) for WT and TIEG1 KO tendon fibers represented in arbitrary units (au) and depending on the scattering (S). The peaks of intensity are labeled with a number corresponding to the order of the peak obtained in the X-ray pattern. The distance between two peaks represent the period D.

Fig. 3.

Ratio of indicated gene expression levels between WT and TIEG1 KO tendon fibroblasts as analyzed by quantitative real-time PCR. Lumican (Lum), laminin beta1-1 (Lamb1-1), and decorin (Dcn) are significantly decreased in TIEG1 KO fibroblasts relative to WT controls. Collagen I (Col1a2) is significantly increased in TIEG1 KO cells. Fibromodulin (Fmod) and lysyl oxydase (Lox) do not exhibit significant differences in expression between these two genotypes. *Significance at the P < 0.05 level.