Patterton D et al. (1995), Transcriptional activation of the matrix metall...

XB-ART-20302

Dev Biol 1995 Jan 01;1671:252-62. doi: 10.1006/dbio.1995.1021.

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Transcriptional activation of the matrix metalloproteinase gene stromelysin-3 coincides with thyroid hormone-induced cell death during frog metamorphosis.

Patterton D , Hayes WP , Shi YB .

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A full-length cDNA was isolated for a thyroid hormone response gene in the metamorphosing frog intestine and shown by sequence analysis to be the frog homolog of the mammalian extracellular matrix metalloproteinase stromelysin-3 (ST3). Northern hybridization indicated that ST3 gene expression is differentially activated in tadpole tissues during metamorphosis. In the small intestine, in situ hybridization localized high levels of ST3 mRNA to fibroblast-like cells during thyroid hormone-induced metamorphosis. ST3mRNA was undetectable in the intestine prior to metamorphosis, while high levels were present at the metamorphic climax. At this time, primary intestinal epithelial cells are known to undergo cell death and replacement by secondary epithelial cells, arguing that ST3 is involved in the modification of the extracellular matrix during apoptosis. ST3mRNA was also expressed at high levels during tadpole tail resorption, but not in premetamorphic tail or developing hindlimb, further supporting a role for ST3 when tissue remodeling is accompanied by large-scale cell death. Premetamorphic tadpoles treated with thyroid hormone showed a similar but compressed time course of ST3 gene regulation, suggesting that thyroid hormone controls ST3 gene expression during metamorphosis. In contrast, during embryogenesis, ST3 was expressed before endogenous thyroid hormone is detectable, indicating that ST3 can also be regulated independently of thyroid hormone. These findings implicate that ST3 participates in the modification of the extracellular matrix during matamorphic apoptosis, but Northern analyses using heterologous probes raise the possibility that additional matrix metalloproteinases may also be involved.

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Species referenced: Xenopus laevis
Genes referenced: gla mmp11 mmp3

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	FIG. 1. Nucleotide and deduced amino acid sequence of a full-length eDNA clone encoding frog strornelysin-3 (STS). The eDNA done contains a 5'-untranslated region of 212 bases, followed by an open reading frame of 477 amino acids and a 1239-base 3'-untranslated region. The wding region can be subdivided into three regions: the prepropeptide (ita lics) and cau.lyticdomain (bold) at theN-terminus of the mature protein and the C-termlnal region. The putative polyadenylation signal sequence is underlined.
	FIG. 2. Frog, human, and mouse ST$s share 64% identity in amino acid sequence. (A) Only amioo acid residues different from the Xenopus ~;equence are shown for the mouse. (Lefebvre et aL, 1992) and human (Basset et aL. 1990) ST3s. Gaps (dashes) were introduced to improve alignment. and two possible cleavage sites (arrowg) predicted for the human prepeptide and the predicted start s ite (arrowhead) of the mature enzyme(BassetetaL,l990) are shown. The propeptide region contains a highly conserved sequence PRCGVPD (underlined) that is charaeteristic of all mammalian MMP propeptides (Alexander and Werb,1991), as well as the (boxed) STS-specific sequence (Lefebvre etaL, 1992). The putative catalytic domain (bracketed) contains the three histidines (asterisks) of the proposed Zn-binding site. Both the catalytic and C-terminal half of the protein show domains identical in frog and mammalian ST9s (hatched bars). (B) The putative ST9 catalytic domain is highly conserved in the frog and mammalian proteins, while I he pre- and propeptides are more dissimilar; numbers indicate the percentage identity between aligned regions of the frog and mouse or human STS protein.

	Fro. 4. High levels of ST9 gene expression were found during metamorphosis in the remodeling intestine and resorbing tall, but not in the developing hindlimb. (A) All lanes have 10 pg of total RNA except for the stage 64 t ail and stage 56 hindlimb lanes, which contain 5 11g. The same blot was probed with either al.2-kbST9cONA fragment or the PR28 control eDNA (see Shi and Brown, l 990). (B) Densitometric quantitat ion of data in (A) shows that STIJ expression is high in the metamor[lhosing intestine (stages 60-62) and tail (stages 62-64) but only very weak in hindlimb at stage 56; levels of ST3 mRNA were normalized to that of tbe PR28 control; to facilitate the graphing of low levels of ST$ mRNA found for hindlimb measured values were multiplied by 50 (X50). Note that the overall decrease in ST3 mRNA at stage 64 when RNA from entire tadpoles were used (Fig. 3) can be explained by the precipitous drop in tail mass betwe~n stages 62 and 64 and t.he simultaneous decrease in intestinal S'/':J mRNA.
	FIG. 5. Thyroid hormone trea t.mentof premetamorpbie tadPOles for the indicated number of days produced a similar but compressed pattern of 81'3 gene expression to that found during natural metamor· phosis. Each lane con tained t() ,.g of total RNA from the intAlstines or tails of stage 56 tadpoles which were treated with 5 nM T3 and probed with the frog STs and control PR28 cDNAs.
	FIG. 6. ln situ hybridization histochemistry using a 35S-labeled cRNA localized STS gene expression to connective tissue cells (C) of tadpole small intestine after thyroid hormone t reatment., whereas STS mRNA was not detectable in untreated tadpoles (not shown). Bl·ight-field and dark-field photomicrographs of cross sections from the small intestines hybridized with an antisense S1'3 probe (A) but not with a sense probe (B) showed loosely clustered ST3-e~pressing cells (arrows) in the stroma of the typhlosole, but not in the intestinal epithelium (E). muscle (M), or lumen (L). (C) At higher magnification, "Mtivated" STS-e:<r>ressing cells appear to be polarized and fibroblast-like (large arrows) and intermixed with "unactivated"' fibroblast-like ce lls (smaller arrows) that do not ex(>ress detectable levels of ST3 mRNA. Note similar cell types appear to express STS in mammalian tissue~ and carcinomas (see Discussion). Cali bration bars arc 200 ,.,m (A. B) and 100 ,.m (C).
	FIG. 7. Hybridization using human eDNA probes suggests that additional matrix metalloproteinases may play a role in frog metamorphosis (see Ma terials and Methods for the rationale and problems associated with using heterologous eDNA probes). (A) Northern blots (like those used in Fig. 4) probed with human eDNA probes suggest expression patterns for the (top) frog stromelysin 1 (STJ) and (bottom) gelatinase A (GLA) genes that are different from frog STS: frog STI-Iike mRNA was expressed throughout development.. whereas in both tail and intestine, frog GLA-Iike mRNA was differentially regulated during metamorphosis. (B) Thyroid hormone treatment of premetamorphic tadpoles showed no effect on the persistent STI-like gene eX]lression. but GLA-like gene expression appeared to be induced. lagging frog ST3 gene activation in the tail and intestine of stage 56 tadpoles treated with T3 for the indicated number of days (compare these findings to the similar blots used in Fig. 6).