Tissue-dependent induction of apoptosis by matrix metalloproteinase stromelysin-3 during amphibian metamorphosis

Abstract

Matrix metalloproteinases (MMPs) are a superfamily of Zn(2+)-dependent proteases that are capable of cleaving the proteinaceous component of the extracellular matrix (ECM). The ECM is a critical medium for cell-cell interactions and can also directly signal cells through cell surface ECM receptors, such as integrins. In addition, many growth factors and signaling molecules are stored in the ECM. Thus, ECM remodeling and/or degradation by MMPs are expected to affect cell fate and behavior during many developmental and pathological processes. Numerous studies have shown that the expression of MMP mRNAs and proteins associates tightly with diverse developmental and pathological processes, such as tumor metastasis and mammary gland involution. In vivo evidence to support the roles of MMPs in these processes has been much harder to get. Here, we will review some of our studies on MMP11, or stromelysin-3, during the thyroid hormone-dependent amphibian metamorphosis, a process that resembles the so-called postembryonic development in mammals (from a few months before to several months after birth in humans when organ growth and maturation take place). Our investigations demonstrate that stromelysin-3 controls apoptosis in different tissues via at least two distinct mechanisms.

Fig. 1

Structure of MMPs. MMPs generally contain five domains. These are the pre-and pro-peptides, catalytic domain, hinge region, and hemopexin domain, respectively. Some exceptions exist. These include matrilysin, which does not have a hemopexin domain, and membrane type MMPs (MT-MMPs), which contain a transmembrane/cytoplasmic (TM) domain at the carboxyl end, and gelatinases, which contain a fibronectin (FN)-like domain. A conserved peptide sequence is present in the pro-peptide where a C residue (underlined) is involved in coordination with the catalytic Zn²⁺ atom in the inactive pro-enzyme. In the catalytic domain, a conserved region contains three H residues (underlined) that coordinate with the catalytic Zn²⁺ atom. Finally, MT-MMPs and ST3 contain the conserved RXKR sequence for furin-dependent intracellular activation of the enzymes.

Fig. 2

(A). Stage-dependent apoptosis in the intestine and tail during Xenopus laevis metamorphosis. Tadpole tail consists of several tissues such as epidermis (Ep), muscle (M), connective tissue (CT), and notochord (Nc), all of which are removed by stage 66. The tails at stages 62-66 are drawn to the same scale to show the resorption, while the tadpoles and intestinal cross-sections (middle) at different stages are not in the same scale in order to highlight the morphological differences. Tadpole small intestine has a single epithelial fold, where much of the connective tissue is localized, while a frog has a multiply folded intestinal epithelium, with elaborate connective tissue and muscle. Asterisks : proliferating cells of adult intestinal epithelium (AE). Open circles: apoptotic cells of larval intestinal epithelium (LE). (B).Organ-dependent temporal regulation of ST3 during Xenopus laevis metamorphosis. The mRNA levels are based on (Patterton and others, 1995) with the expression level in tail at stage 64 set to 100.

Fig. 2

(A). Stage-dependent apoptosis in the intestine and tail during Xenopus laevis metamorphosis. Tadpole tail consists of several tissues such as epidermis (Ep), muscle (M), connective tissue (CT), and notochord (Nc), all of which are removed by stage 66. The tails at stages 62-66 are drawn to the same scale to show the resorption, while the tadpoles and intestinal cross-sections (middle) at different stages are not in the same scale in order to highlight the morphological differences. Tadpole small intestine has a single epithelial fold, where much of the connective tissue is localized, while a frog has a multiply folded intestinal epithelium, with elaborate connective tissue and muscle. Asterisks : proliferating cells of adult intestinal epithelium (AE). Open circles: apoptotic cells of larval intestinal epithelium (LE). (B).Organ-dependent temporal regulation of ST3 during Xenopus laevis metamorphosis. The mRNA levels are based on (Patterton and others, 1995) with the expression level in tail at stage 64 set to 100.

Fig. 3

Transgenic overexpression of ST3 preferentially induces apoptosis in the muscles of the tadpole tail. Premetamorphic transgenic (B) and sibling wild type (A) tadpoles were subjected to daily heat shock treatment to induce transgene expression. After 4 days the tadpoles were sacrificed and the tail was isolated for TUNEL assay to detect apoptotic cells. M: muscle, Ep: epidermis, Mc: melanocytes; arrows: apoptotic cells. (C) Quantification of the apoptotic cells in the muscle or epidermal region per an arbitrarily defined unit area sections of the tail of wild and transgenic tadpoles. Note that ST3 over expression led to an increase in apoptosis in both the muscle and epidermal regions but significantly only in the muscle region. *, P value ≤0.05. For more details, see (Mathew and others, 2009).

Fig. 4

High levels of ST3 expression correlate with cell death in the metamorphosing tail. (A). ST3 mRNA attains high levels by stage 62, when tail length reduction is to begin during natural metamorphosis, and reaches even higher levels by stage 63, when the tail is resorbed to half of its original length. (B). Apoptotic cells per defined unit area of the tail at stage 61 and stage 63. M: muscles; Ep: epidermis. For more details, see (Mathew and others, 2009).

Fig. 5

(A). LR cleavage is present at stages 62 and 63 when ST3 expression is very high in the tail. Total protein was isolated from tail of tadpoles at various stages. The proteins were subjected to western blotting with anti-Xenopus LR antibody. For more details, see (Mathew and others, 2009). (B). LR is cleaved in the tail and intestine at the climax of metamorphosis as well as in the intestine but not tail of premetamorphic transgenic tadpoles overexpressing ST3. Total protein was isolated from tail or intestine of heat shock (HS)-treated transgenic (TG) or wild type premetamorphic animals or naturally metamorphosing tadpoles at stage 62 or 63. The proteins were subjected-to western blotting with anti-Xenopus LR antibody. Note that LR cleavage was observed in stage 63 tail and stage 62 intestine as well as in the intestine of transgenic tadpoles overexpressing ST3. No cleavage was detected in transgenic tail. (C). LR is cleaved in the epidermis but not the muscles of the tail at the climax of metamorphosis. Total protein was isolated from the tail of stage 63 naturally metamorphosing tadpoles or isolated epidermis and muscles of the stage 63 tail. The proteins were subjected to western blotting with anti-Xenopus LR antibody. Note that the LR in the muscle lane was from the connective tissue surrounding the muscle cells. The black arrowhead indicates full-length LR. The gray arrowheads indicate likely non-specific cleavage products of LR. The open and closed circles indicate the expected ST3 cleavage products. For more details, see (Mathew and others, 2009).