Regulation of extracellular matrix remodeling and cell fate determination by matrix metalloproteinase stromelysin-3 during thyroid hormone-dependent post-embryonic development

Abstract

Interactions between cells and extracellular matrix (ECM), in particular the basement membrane (BM), are fundamentally important for the regulation of a wide variety of physiological and pathological processes. Matrix metalloproteinases (MMP) play critical roles in ECM remodeling and/or regulation of cell-ECM interactions because of their ability to cleave protein components of the ECM. Of particular interest among MMP is stromelysin-3 (ST3), which was first isolated from a human breast cancer and also shown to be correlated with apoptosis during development and invasion of tumor cells in mammals. We have been using intestinal remodeling during thyroid hormone (TH)-dependent amphibian metamorphosis as a model to study the role of ST3 during post-embryonic tissue remodeling and organ development in vertebrates. This process involves complete degeneration of the tadpole or larval epithelium through apoptosis and de novo development of the adult epithelium. Here, we will first summarize expression studies by us and others showing a tight spatial and temporal correlation of the expression of ST3 mRNA and protein with larval cell death and adult tissue development. We will then review in vitro and in vivo data supporting a critical role of ST3 in TH-induced larval epithelial cell death and ECM remodeling. We will further discuss the potential mechanisms of ST3 function during metamorphosis and its broader implications.

Fig. 1

Structure of ST3. (A) ST3 protein. Like most MMPs, ST3 contains four domains. These are the pre- and pro-peptides, and catalytic and hemopexin-like domains from N- to C-terminus, respectively. A conserved peptide sequence is present in the propeptide where a C residue (underlined) is involved in coordination with the catalytic Zn atom in the inactive proenzyme. In the catalytic domain, a conserved region contains three H residues (underlined) that coordinate with the catalytic Zn atom. Like membrane type-MMPs, ST3 contains an RXKR sequence for furin-dependent intracellular activation. (B) ST3 gene. The hinge region and the hemopexin-like domain of ST3 are encoded by only 4 exons instead of 6 exons as in other MMPs (Anglard et al., 1995; Li et al., 1998; Wei & Shi, 2005). The individual exons are shown as bars. The dotted bars indicate the coding region and the open bars represent the 5′- and 3′-UTRs. The individual domains of the coding region are indicated on the top.

Fig. 2

Intestinal metamorphosis is accompanied by ECM remodeling during Xenopus laevis metamorphosis. Top: Animals at the onset of metamorphic climax (stage 57), metamorphic climax (stage 60), and the end of metamorphosis (stage 66) (Nieuwkoop & Faber, 1956). Middle: Cross-sections of the intestine stained with pyronin-Y (red staining) and methyl green (blue staining) to show the morphology. The strong pyronin-Y signals observed in the clusters of cells (arrows) at metamorphic climax indicate islets of proliferating adult epithelial cells. Bottom: The ECM that separates epithelium and connective tissue is thin before and after metamorphosis (arrow heads), and becomes thicker at the metamorphic climax (double-headed arrow). LE: larval/tadpole epithelium, AE: adult epithelium, CT: connective tissue, MU: muscles, I: islets (proliferating adult epithelial cells), bl: basal lamina or basement membrane.

Fig. 3

Transgenic overexpression of ST3 leads to basal lamina alteration and establishment of direct contacts between epithelial cells and fibroblasts. The intestines from wild type tadpoles at metamorphic climax (stage 61) and stage 54 wild type or transgenic tadpoles after 4 days of heat shock treatment to induce the expression of transgenic ST3 were isolated and analyzed under an electron microscope (the transgene was under the control of a heat shock inducible promoter). (A and a). A thin and continuous basal lamina (bl, arrow heads) separates the epithelium from the connective tissue in the intestine of stage 54 wild type tadpoles with heat shock treatment, similar to wild type animals without heat shock. Panel a shows the basal lamina in the boxed region of A at a higher magnification. (B, b). The basal lamina becomes amorphous or absent in the intestine in transgenic tadpoles expressing ST3 after heat shock treatment. Furthermore, overexpression of ST3 led to the activation of fibroblasts just beneath the epithelium, as reflected by the presence of well-developed rough endoplasmic reticulum (rER) in these cells. Panel b shows the boxed region in panel B revealing the lack of basal lamina in this region of the ST3 transgenic tadpole. (C). Epithelial cell-fibroblast contacts are present in tadpoles overexpressing ST3 but not in wild type at stage 54. (D). The basal lamina (double-headed arrow) becomes much thicker at the climax of metamorphosis (stage 61), when the fibroblasts are also activated as shown by the presence of well-developed rER. Ep: epithelial cell; Fb: fibroblast, bl: basal lamina; rER: rough endoplasmic reticulum. Bar: 1 μm. See (Fu et al., 2005) for more details.

Fig. 4

ST3 cleaves both human and Xenopus LR between the transmembrane domain (TM) and laminin binding sequence (LB). (A) ST3 cleaves human LR at the same sites as in Xenopus LR. ³⁵S labeled full-length human and Xenopus LR were synthesized in vitro and digested with purified ST3 catalytic domain in vitro. Note that N-terminal cleavage products (a-N and b-N) of human and Xenopus LR are of the same sizes whereas the full-length protein and C-terminal cleavage products of human LR were smaller than the corresponding ones in Xenopus due to length divergence. (B) Schematic diagram of LR localization on cell surface with the two ST3 cleavage sites indicated by two arrows (see (Amano et al., 2005b) for details).

Fig. 5

ST3 degrades LR in the intestine during metamorphosis. LR is degraded in the intestine at the climax of metamorphosis and in the intestine of premetamorphic transgenic tadpoles overexpressing ST3. Transgenic animals (T) and non-transgenic siblings (W) at stage 56 were treated with or without heat shocked daily in the same tank. Two or three days later, the intestines were isolated. Total protein was isolated from these intestines as well as from the intestines of tadpoles at stage 57, prior to intestinal metamorphosis, or stage 61, at the climax of intestinal metamorphosis. The proteins were subjected to Western blotting with anti-Xenopus LR antibody. The open arrow indicates full length LR. The triangles, and solid and open circles indicate degradation products. The larger bands (triangles) were occasionally observed in different sample preparations in tadpoles at different stages but the bands indicated by the circles were found at much higher levels in metamorphosing intestine or in animals expressing transgenic ST3 (see (Amano et al., 2005a) for details).

Fig. 6

Tissue-dependent spatial and temporal regulation of LR expression during intestinal metamorphosis. Intestinal cross sections from tadpoles at indicated stages were immunostained with anti-Xenopus LR antibody. L, lumen; LE, larval epithelium; AE adult epithelium; CT, connective tissue. The greenish brown labeling is due to LR antibody staining and the nuclei have blue staining (see (Amano et al., 2005a) for details).