Expression in SPARC-null mice of collagen type I lacking the globular domain of the α1(I) N-propeptide results in abdominal hernias and loss of dermal collagen

Abstract

The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2 months of life with an approximate 20% penetrance (~35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9 nm versus exon 2-deleted: 88.2 nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2 nm, SPARC-null/exon 2-deleted: 40.8 nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.

Figure 1

A fraction of SPARC-null/exon 2-deleted mice develop hernias within the first two months of age. White arrows denote an abdominal protrusion in A and sites of herniation in B and C.

Figure 2

Representative sections of skin from age-matched mice at 3 months stained with hematoxylin and eosin: A) WT, B) Exon 2-deleted, C) SPARC-null, and D) SPARC-null/exon 2-deleted mice (D). Scale bar in A equals 1.0 mm.

Figure 3

Representative sections of skin stained with PSR and viewed under polarized light. A) WT, B) Exon 2-deleted, C) SPARC-null, and D) SPARC-null/exon 2-deleted. Scale bar in A equals 50 μm.

Figure 4

Representative EM images from Exon 2-deleted (A and C) and SPARC-null/Exon 2-deleted dermal sections. Bar in B equals 2 μm for panels A and B. Bar in D equals 500 nm for panels C and D. E) Collagen fibril diameter distribution for the four genotypes. WT (black bars), Exon 2-deleted (light gray bars), SPARC-null (dark gray bars), SPARC-null/exon 2-deleted (double deleted, white bars).

Figure 5

A) Dry weight of skin biopsies and B) hydroxyproline analysis of 8 mm punch biopsies. Four animals per genotype and 2 biopsies per animal contributed to the analysis. WT (black bars), Exon 2-deleted (light gray bars), SPARC-null (dark gray bars), SPARC-null/exon 2-deleted (double deleted, white bars). Error bars represent standard error of the mean. # p<0.05 versus WT, * p<0.05 versus Exon 2-deleted, ^ p<0.05 versus SPARC-null.

Figure 6

Acetic acid extraction of collagen from skin. Top panel: Coomassie-blue stained SDS-PAGE of collagen extracted in acetic acid in equal weight per volume. Equal volumes of acetic extracts were loaded. Bottom panel: samples shown in top panel analyzed by immunoblot analysis using anti-murine collagen I antibody. Lane 1: WT; Lane 2: exon 2-deleted, Lane 3: SPARC-null, Lane 4: SPARC-null/exon 2-deleted. MW: molecular weight markers.