MECHANISM AND FUNCTION OF TR DURING DEVELOPMENT. We have proposed a dual function model for TR during frog development. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes in vivo. In premetamorphic tadpoles, they repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. When TH is present either from endogenous synthesis during development or exogenous addition to the raring water of premetamorphic tadpoles, TR/RXR heterodimers activate TH-inducible genes to initiate metamorphosis. We have shown that TR is both necessary and sufficient for the metamorphic effects of TH. Thus metamorphosis provides the first example where TR is shown to mediate directly and sufficiently the developmental effects of TH. To investigate whether unliganded TR also plays a role in premetamorphic development by repressing TH target gene expression, as predicted by our dual function model, we designed a dominant negative form of the TR-binding corepressor N-CoR (nuclear receptor corepressor) (dnN-CoR), which contains only the receptor interacting domain of N-CoR. We overexpressed dnN-CoR under the control of a heat shock inducible promoter in tadpoles through transgenesis. We observed significant derepression of TH-response genes in transgenic animals. More importantly, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. These data thus provide in vivo evidence for the presence and a role of unliganded TR-induced gene repression in physiological settings and strongly support our dual function model. ? ? ROLES OF COFACTORS IN GENE REGULATION BY TR. TR regulates gene transcription by recruiting cofactors to target genes. In the presence of TH, TR can bind to coactivators while the unliganded TR binds to corepressors. Many biochemical and molecular studies have been done on such cofactors. On the other hand, much less is known about whether and how they participate in gene regulation by TR in different biological processes in vivo. Our studies with the dnN-CoR as summarized above have now demonstrated in vivo a role of corepressor complexes in gene repression by unliganded TR during premetamorphic tadpole development. On the activator side, we have shown that Xenopus coactivator SRC3 (steroid receptor coactivator 3) is recruited in a gene- and tissue-dependent manner to target genes by TR. Transgenic expression of a dominant negative form of SRC3 (F-dnSRC3) inhibited essentially all aspects of metamorphosis. Our studies thus demonstrate that coactivator recruitment, aside from corepressor release, is required for TH function in development and further provide the first example where a specific coactivator-dependent gene regulation pathway by a nuclear receptor has been shown to underlie specific developmental events. While these studies demonstrated the importance of coactivators in TR function during metamorphosis, there are numerous TR-binding coactivators. The F-dnSRC3 would block the binding to TR of not only SRC3 (and the highly related cofactors, SRC1, 2) but also other coactivators. Thus, it remains unclear whether SRCs (SRC1-3) play an essential role in metamorphosis. To investigate this possibility, we analyzed the histone acetyltransferase p300, which is a component of the SRC-coactivator complexes. We showed that p300 was recruited to TH-responsive promoters, implicating a role of p300 in TR function. Furthermore, we generated a dominant negative form of p300, F-dnp300, which contained only the SRC-interacting domain of p300 and thus would interfere with the function of only SRCs but not other TR-binding coactivators. Transgenic tadpoles overexpressing F-dnp300 also displayed arrested or delayed metamorphosis and had reduced expression of TH-responsive genes. Our results thus suggest that p300 and SRC proteins are part of the same critical coactivator complexes in vivo during postembryonic development. ? ? REGULATION AND FUNCTION OF THE MATRIX METALLOPROTEINASE (MMP) STROMELYSIN-3 (ST3) DURING TH-INDUCED TISSUE REMODELING. We have previously identified several TH-response genes encoding MMPs during intestinal metamorphosis. MMPs are Zn2+-dependent proteases capable of cleaving different proteins of the ECM (extracellular matrix). The ECM not only provides the essential physical support within an organism but also influences cell fate during developmental and pathological processes. Upregulation of MMP genes has been observed in many developmental processes such as limb morphogenesis and in diverse pathological conditions such as arthritis, wound healing, and cancer metastasis. However, it has been difficult to investigate the roles of MMPs in mammals due to the lack of good models and the relatively subtle phenotypes in limited tissues in knockout mice. Frog metamorphosis affects essentially all organs in a tadpole within a short developmental period and thus offers an excellent opportunity to investigate the in vivo functions of MMPs and the associated mechanisms during development. Expression and organ culture studies have led us to propose that the MMP ST3 is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior, in the metamorphosing intestine. Transgenic studies have shown that precocious overexpression of ST3 but not a catalytically inactive mutant leads to premature apoptosis in the intestinal epithelium of premetamorphic tadpoles. Toward understanding the mechanism by which ST3 affects tissue remodeling, we have used yeast two-hybrid screen to isolate the 37 kd laminin receptor (LR), a cell surface receptor for the ECM protein laminin, as a substrate. LR binds to ST3 and can be cleaved by ST3 at two sites both in vitro and in vivo in the extracellular domain between the transmembrane domain and laminin binding sequence, suggesting that LR cleavage by ST3 will alter cell-ECM interaction. Interestingly, ST3 cleavage sites in LR are conserved in human LR and high levels of LR are known to be expressed in tumor cells, which are often surrounded by fibroblasts expressing ST3. Thus, LR may be a conserved substrate of ST3 and its cleavage by ST3 may alter cell-ECM interactions, thereby mediating the effects of ST3 on cell fate and behavior during development and pathogenesis.? ? A COOPERATIVE ROLE OF GELATINASE A (GELA) AND MEMBRANE TYPE-1-MMP (MT1-MMP) DURING XENOPUS LAEVIS DEVELOPMENT. In addition to ST3, several other MMP genes are also activated during amphibian metamorphosis. This is consistent with the complex nature of the ECM, which would require multiple enzymes for its remodeling and degradation during metamorphosis. Interestingly, unlike ST3, the MMP GelA appeared to be a late TH response gene in both the tail and intestine during metamorphosis, implicating different roles for this MMP compared to those for ST3. We have now analyzed its detailed spatial and temporal expression profiles. In addition, we cloned and analyzed Xenopus MT1-MMP, whose mammalian homolog has been shown to participate in the activation of pro-GelA to the active or mature GelA lacking the pro-peptide. We showed that both MT1-MMP and GelA were coordinately upregulated and coexpressed in the intestine and tail during metamorphosis, suggesting that MT1-MMP and GelA function together in ECM degradation or remodeling during metamorphosis. Furthermore, we used Xenopus embryogenesis as a model to show that they interacted with each other in vivo and that overexpression of MT1-MMP and GelA together in Xenopus embryos led to the activation of pro-GelA and a cooperative effect on embryonic development. Thus, our studies support a cooperative role of these MMPs during development, at least in part through the activation of pro-GelA by MT1-MMP.
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