Distinct functions for the catalytic and domains of a Drosophila matrix

Bernadette M. Glasheen, Aashish T. Kabra1, and Andrea Page-McCaw2

Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180

Edited by Terry L. Orr-Weaver, Massachusetts Institute of Technology, Cambridge, MA, and approved December 29, 2008 (received for review April 29, 2008)

Human matrix (MMPs) are believed to contrib- Mmp1 allele Mmp1 predicted protein ute to tumor progression. Therapies based on inhibiting the cat- alytic domain of MMPs have been unsuccessful, but these studies wild-type ss pro catalytic hemopexin f1 raise the question of whether other MMP domains might be f2 appropriate targets. The genetic dissection of domain function has ss pro catalytic hemopexin been stymied in mouse because there are 24 related and partially ss pro catalytic hemopexin PC redundant MMP in the mouse genome. Here, we present a genetic dissection of the functions of the hemopexin and catalytic 2 (excision) ss pro domains of a canonical MMP in Drosophila melanogaster,an organism with only 2 MMPs that function nonredundantly. We Q112* (genetic null) ss pro catalytic compare the phenotypes of Mmp1 null alleles with alleles that have specific hemopexin domain lesions, and we also examine phenotypes of dominant-negative mutants. We find that, although Q273* (hemopexin deleted) ss pro catalytic the catalytic domain appears to be required for all MMP functions including remodeling of the tracheal system, W439* ss pro catalytic hemopexin the hemopexin domain is required specifically for tissue invasion (hemopexin truncated) events later in metamorphosis but not for tracheal remodeling. Thus, we find that this MMP hemopexin domain has an apparent Mmp1 transgene Mmp1 predicted protein specialization for tissue invasion events, a finding with potential

implications for inhibitor therapies. DN pro-pex ss pro hemopexin (dominant negative) cancer ͉ genetics ͉ tissue remodeling A DN E225A ss pro catalytic hemopexin (dominant negative)

atrix metalloproteinases (MMPs) are a family of BIOLOGY Fig. 1. Predicted protein products of Mmp1 alleles and transgenes. (Upper)

Mrequired for tissue remodeling, and their expression is Predicted products from alleles at the Mmp1 genomic locus. The wild-type DEVELOPMENTAL up-regulated in tumors and inflamed tissue. As that protein has the typical domain structure of a secreted MMP, including a signal cleave extracellular matrix, MMPs have the potential to break sequence (ss), an inhibitory pro domain (pro), a catalytic domain (cat) (the down tissues, remove physical barriers, and liberate signaling catalytic core is shown as a v-shaped indentation), a flexible hinge domain molecules. All of these processes occur during tissue remodeling (shown as a black wavy line), and a hemopexin domain (pex) containing 4 and during tumor progression. MMP mutant phenotypes in mice hemopexin loops (each light gray). We have identified cDNAs for 2 splice forms and flies demonstrate that MMPs are required for tissue remod- of Mmp1 as shown, form 1 (f1, known as PD in Flybase) and form 2 (f2). Flybase eling (reviewed in ref. 1), and many lines of evidence implicate predicts an additional splice form PC, shown for completeness. The dashed line MMPs in promoting tumor progression including clinical data, over the catalytic domain denotes the recombinant fragment used for gen- erating anti-catalytic monoclonal antibodies. Allele 2, generated by P- mouse tumor studies, cell culture studies, and substrate analysis element excision (18), deletes most of the coding sequence. Q112*, Q273*, (reviewed in refs. 2 and 3). Thus, MMPs are considered potential and W439*, all recovered in an EMS mutagenesis screen (18), each contain a pharmaceutical targets for cancer therapies, although some nonsense mutation causing premature termination as shown. (Lower) Mmp1 MMPs may be important for inhibiting tumor progression transgenes with dominant-negative activity, used under UAS transcriptional (reviewed in ref. 3). control. DN Pro-pex is a deletion construct lacking the entire catalytic domain. In the 1990s, the pharmaceutical industry performed clinical DN E225A is a point mutant that ablates the conserved E225 at the catalytic trials to test several MMP inhibitors that had been effective in core, rendering the catalytic domain nonfunctional. preventing tumor progression in mouse (4–7). These compounds were designed to inhibit MMP catalysis at the (8). teinases) can reversibly occupy the active site of the catalytic Unfortunately, in patients MMP inhibitors caused musculoskel- domain and thus regulate its activity (10). The pro domain acts etal pain and inflammation, which decreased the tolerated dose as a negative regulator of catalytic activity by occupying the possibly below effective levels (9). From these studies, it can be concluded that the broad-spectrum inhibition of MMP catalysis is not a workable strategy for patient therapies as MMP catalysis Author contributions: A.P.-M. designed research; B.M.G., A.T.K., and A.P.-M. performed is required in normal physiology. Other domains may be more research; B.M.G., A.T.K., and A.P.-M. analyzed data; and A.P.-M. wrote the paper. appropriate inhibitor targets, and this possibility raises a ques- The authors declare no conflict of interest. tion about MMP structure/function: Do the domains participate This article is a PNAS Direct Submission. equally in different biological processes, or do some domains Freely available online through the PNAS open access option. participate in some processes and not others? 1Present address: Neuroscience Institute and Department of Neurology, Albany Medical MMPs contain 3 highly conserved domains: the pro, catalytic, College, Albany, NY 12205. and hemopexin domains (see Fig. 1). The catalytic domain 2To whom correspondence should be addressed. E-mail: [email protected]. mediates proteolysis of substrates and is often expressed in This article contains supporting information online at www.pnas.org/cgi/content/full/ isolation for in vitro proteolysis assays. Endogenous MMP 0804171106/DCSupplemental. protein inhibitors called TIMPs (tissue inhibitors of metallopro- © 2009 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804171106 PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2659–2664 Downloaded by guest on September 26, 2021 active site; it is removed for activation. The hemopexin animals, Table 1), these were much lower than levels in null domain, which is connected to the catalytic domain by a flexible animals (Ϸ45%), and we did not observe the taut stretched hinge, is a beta-propeller structure comprised of 4 repeating dorsal trunks typical of the null animals. It is likely that the loops, each of which is homologous to the blood protein he- tracheal breaks in the hemopexin mutant larvae are caused by mopexin. This domain is believed to mediate protein–protein the overall reduction in Mmp1 protein (see below) rather than interactions and to contribute to substrate specificity (11–15). by the lack of the hemopexin domain. Animals mutant for either One method of assessing the functions of different domains is hemopexin allele Q273*orW439* frequently survived to meta- through genetic analysis of mutants. However, a complication of morphosis, when they died with body malformations associated MMP genetic analysis in mouse models is that there are 24 MMP with a failure of disc eversion (Table 1 and Fig. 3). Transhet- genes that exhibit partial redundancy (reviewed in ref. 1). In erozygotes of the genotype Q273*/W439* also had normal larval contrast, the fruitfly Drosophila melanogaster has only 2 MMP dorsal trunks with infrequent breaks, survived to pupariation, genes, Mmp1 and Mmp2, and each has the conserved domain and sometimes failed to evert imaginal discs during metamor- structure typical of mammalian MMPs (16–18). Each Drosophila phosis. Early in disc eversion, imaginal disc peripodial and stalk MMP is required for viability, participating in different aspects cells traverse 2 layers of basement membrane to invade the larval of postembryonic tissue remodeling. Mmp1 is required for larval body wall (23). Srivastava et al. have demonstrated that in tracheal elongation and for tissue invasion during disc eversion animals compromised for Mmp1 function, the normal invasion of during metamorphosis (18, 19); Mmp2 is required for histolysis basement membranes by disc epithelia fails, leading to failures of and epithelial fusion during metamorphosis, and it is not re- disc eversion (19). The observation that both Mmp1 hemopexin quired in the larval tracheal system (18). Interestingly, both mutants fail to evert discs indicates that the hemopexin domain Drosophila MMPs have been demonstrated by 3 groups to be is required selectively for developmental tissue invasion but not required for tumor invasion using 2 different Drosophila tumor for remodeling the tracheal tubes (Table 1). models (19–22), suggesting a conservation of pathological and To understand these alleles better, we examined Mmp1 wild- physiological MMP function in Drosophila and humans. Here, we type and mutant protein mobility and expression levels by analyze the phenotypes of Mmp1 mutants disrupted for the Western blot analysis with anti-Mmp1-catalytic-domain mono- hemopexin domain and compare them to Mmp1 null mutants. clonal antibodies (Fig. 4; comparisons of the individual mono- We find that the Drosophila Mmp1 catalytic domain is required clonal antibodies are shown in supporting information (SI) Fig. for tracheal tissue remodeling whereas the hemopexin domain is S1). Lysates of wild-type embryos and larvae showed several not required for tracheal tissue remodeling, but rather is re- Mmp1-specific bands, most prominently at 64, 52, and 46 kDa, quired specifically for tissue invasion events. with a larger band at 74 kDa observed prominently in embryos but only faintly in larvae. No bands were observed in a control Results extract made from Mmp1 null embryos and larvae, confirming In ref. 18, we reported that Mmp1 null mutant larvae failed to the identity of the bands as Mmp1. The expected size of Mmp1 remodel their tracheae, the branched respiratory organ. This is unclear because of different splice isoforms: We previously conclusion is based on the analysis of the two null alleles shown identified cDNAs for 2 alternatively spliced proteins we called in Fig. 1: Mmp12 is a P-element excision allele lacking nearly all Mmp1.f1 (aka Mmp1-PD at Flybase, FBpp0271772) and of the Mmp1 ORF; Mmp1Q112* is an EMS-induced mutation Mmp1.f2 (18); another isoform called Mmp1-PC is reported at coding for a protein prematurely truncated in the catalytic Flybase FBpp0271771. No functional differences are known for domain (18). In null mutant animals (both homozygotes and the splice forms. All of these isoforms contain the full catalytic transheterozygotes), the tracheal tubes appear to form normally and hemopexin domains and differ only in their carboxy- during embryogenesis, but the tubes cannot elongate as the terminal ends (see Fig. 1); these are predicted to encode proteins animal grows during larval instars. Instead, the mutant tracheal of 65 (PC), 59 (f1 or PD), and 57 (f2) kDa. Additionally, any tubes are pulled tighter along the length of the animal, appearing expressed Mmp1 isoform is expected to be activated by removal increasingly taut until they frequently rip, with breaks appearing of the 108-aa autoinhibitory Pro domain, resulting in products in the tubes (Table 1 and Fig. 2). Null alleles are homozygous reduced in size by Ϸ11.6 kDa. Thus, the 74 kDa band in embryos lethal, and mutants die as second and third instar larvae (18). is substantially larger than expected, perhaps owing to posttrans- The null phenotype demonstrates that Mmp1 is required for lational modification. tracheal elongation. We observed protein bands for both hemopexin mutants. In an EMS screen to identify new alleles of Drosophila Mmp1, Mmp1Q273* protein is predicted to be 30 kDa (as the nonsense we identified alleles that specifically affect the hemopexin mutation affects all splice forms). In embryo lysates, 2 faint domain (18). The first allele, Q273*, has a nonsense mutation at bands were observed at 35 kDa and 22 kDa, indicating possible Q273, leading to a premature truncation of the protein in the posttranslational modification. The low level of Mmp1Q273* flexible hinge domain so that the predicted protein lacks the protein was consistent with our previous genetic analysis, which entire hemopexin domain (Fig. 1). Interestingly, the Q273* showed that Q273* is a hypomorphic allele whose severity predicted protein mimics mammalian recombinant MMPs that increases when in trans to a null allele (18). Thus, the Q273* lack a hemopexin domain, engineered to assay proteolysis in allele can be classified as a hypomorph with respect to the vitro. The second allele, W439*, has a nonsense mutation at catalytic domain, and as a null with respect to the hemopexin W439, which causes a premature truncation of the hemopexin domain, because this domain is absent from the product. domain in the third of the 4 hemopexin loops (Fig. 1). These Despite several attempts, the level of mutant protein in second mutant alleles afford the opportunity to dissect the function of instar larvae was below our detection threshold, although phe- the hemopexin domain in vivo. notypic analysis indicates that some protein is present in Q273* Tracheal breaks were infrequent in the Q273* and W439* larvae. It is remarkable that tracheal elongation, a process that homozygous larvae (see Table 1). Instead, these hemopexin requires Mmp1, can still occur with these undetectably low levels mutants displayed fairly normal tracheae, which were able to of Mmp1 protein in second instar larvae. Most importantly, the grow as the animal elongated (Fig. 2). This observation was Q273* allele demonstrates that the hemopexin domain is dis- surprising, because we expected that hemopexin-mediated sub- pensable for tracheal elongation. strate recognition would be required for all Mmp1-mediated For the W439* mutant protein, the zymogen is expected to be functions, including tracheal elongation. Although we did ob- 49 kDa, and 2 distinct bands were observed at 50 and 38 kDa with serve slightly elevated levels of tracheal breaks (in Ϸ7% of a fainter band at 31 kDa (Fig. 4B), indicating that this mutant

2660 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804171106 Glasheen et al. Downloaded by guest on September 26, 2021 Table 1. Phenotypes of catalytic and hemopexin-disrupted Mmp1 mutants Broken dorsal Survive to trunks, pupariation, Disc eversion lethal Genotype Nature of allele %(n*) %(n†) phenotypes (n)

w1118 Control 0 (100) 100% (100) All viable, wings and head everted (100) Mmp1 Q273*/CyO Control 2.5% (119) ND ND Mmp12 Null 45% (105) 0 (65) NA Mmp1Q112* Null 43% (54) 0 (46) NA Mmp1Q112*/2 Null trans-het 44% (63) ND NA

Mmp1Q273* Hemopexin deletion 6.1% (147) 59% (206) 15/25 wings and head everted 10/25 wings everted, no head

Mmp1W439* Hemopexin truncation 7.0% (100) 23% (100) All have no wings or head everted (14)

Mmp1Q273*/W439* Hemopexin trans-het 6.1% (115) 78% (115) 21/24 wings and head everted 4/24 wings everted, no head

Mmp2W307*/Df Mmp2 null trans-het 0 (107) 94% (107) 11/25 wings and head everted 14/25 wings everted, no head

tubP Ͼ DN Pro-pex.f1 (1) Ubiquitous hemopexin-interfering 0.6% (163) 96% (70) 4/25 wings and head everted 9/25 wings everted, no head 12/25 no wings or head everted

tubP Ͼ DN Pro-pex.f1 (9) Ubiquitous hemopexin-interfering 0 (73) 97% (73) 10/26 wings and head everted 16/26 no wings or head everted

tubP Ͼ DN Pro-pex.f1 (1), 2 copies ubiquitous hemopexin- 2.7% (108) 96% (98) 22/25 wings everted, no head‡ DN Pro-pex.f1 (9) interfering 3/25 no wings or head everted

Mmp1Q273*/W439*; Hemopexin trans-het expressing 1.8% (57) ND 5/25 wings and head everted Ͼ tubP DN Pro-pex.f1 (1) hemopexin interfering 1/25 one wing and head everted 14/25 wings everted, no head 5/25 one wing everted, no head

tubP Ͼ DN E225A.f1 Ubiquitous dominant negative 0.9% (110) 88% (110) 1/25 wings and head everted

24/25 wings everted, no head BIOLOGY DEVELOPMENTAL tubP Ͼ DN E225A.f2 Ubiquitous dominant negative 1.0% (96) 85% (96) All have wings everted, no head (26)

Mmp1Q273*/W439*; tubP Ͼ DN Hemopexin trans-het expressing 14.9% (47) ND 6/26 wings everted, no head E225A.f1 dominant negative 20/26 no wings or head everted

Btl Ͼ DN Pro-pex.f1 (9) Hemopexin-interfering in tracheae 0 (113) ND ND Btl Ͼ DN E225A.f1 Dominant negative in tracheae 0.9% (107) ND ND Btl Ͼ DN E225A.f2 Dominant negative in tracheae 0 (106) ND ND Btl Ͼ Timp Catalytic inhibition in tracheae 61% (110) 63% (100) ND

n, number of animals examined; ND, not done; NA, not applicable. *Observed in 3rd instar larvae. †First instar survival to pupariation was measured. ‡These animals were overall considerably less developed compared with each single insertion of DN Pro-pex.

protein is the expected size within error of our observation. We we constructed a hemopexin-interfering Mmp1 transgene. This have established that the W439* allele is a hypomorph, because mutant, DN Pro-pex, removed the entire catalytic domain coding more animals pupariate in homozygotes than in W439*/2 ani- region but retained a full-length hemopexin domain (Fig. 1). It mals; but W439* is a stronger allele than Q273*, as shown by its was predicted to behave as a dominant negative by interfering earlier lethality (both in homozygotes and in trans to allele 2) with wild-type Mmp1 in binding protein partners via the he- (18). We were surprised that so much more protein was evident mopexin domain. Its dominant-negative nature was experimen- for W439* than for the weaker Q273* mutant, suggesting that tally confirmed. We showed that the misexpression of Mmp1 although W439* mutant message is translated, most of the with a dpp-GAL4 driver led to a phenotype of short, thick, and protein is not functional—perhaps it does not fold correctly, is easily broken thoracic bristles, and that this bristle phenotype not exported from the cell, and therefore would not be not was suppressed by the coexpression of UAS-Timp (18). DN posttranslationally modified. For both alleles then, the reduction Pro-pex was also able to suppress thoracic bristle phenotypes in protein levels, or specifically in catalytic domain levels, may caused by dpp-GAL4 driving Mmp1 expression in adult flies (Fig. explain the partial larval lethality and the infrequent tracheal 5). This suppression was not caused by the dilution of GAL4 breaks observed in the two hemopexin mutants. spread between 2 UAS elements, because the presence of We wanted to independently assess whether the hemopexin UAS-GFP had no effect on the dppϾMmp1 bristle phenotype domain participates in larval tracheal remodeling, without the (Fig. 5B). Because it phenocopied the endogenous Mmp1 in- complications of reduced catalytic domain levels in these he- hibitor Timp, we conclude that Mmp1DN Pro-pex acts in a mopexin mutants. To disrupt the hemopexin-domain function, dominant-negative fashion.

Glasheen et al. PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2661 Downloaded by guest on September 26, 2021 ABC D

wt Mmp12 (null) Mmp12 (null) btl>Timp E F G H

Mmp1Q273* Mmp1W439* tubP>Mmp1DN pro-pex.f1 tubP>Mmp1DN E225A.f1

Fig. 2. Tracheal phenotypes in larvae with nonfunctional Mmp1 catalytic and hemopexin domains. Reflected light images of the posterior ends of third instar larvae are shown, all in dorsal view with anterior on the left. (A) Wild-type. (B) Mmp12 null, displaying broken dorsal trunks (arrows show breaks). (C) Enlargement of box shown in B, with the broken dorsal trunk tip marked with an arrow. (D) Larva expressing the MMP catalytic inhibitor Timp in the tracheae using the btl-GAL4 driver, displaying broken dorsal trunks (arrows show breaks). (E) Mmp1Q273* larva, lacking the Mmp1 hemopexin domain, with apparently normal dorsal trunks. (F) Mmp1W439* larva, lacking part of the Mmp1 hemopexin domain, also displaying relatively normal dorsal trunks. (G) Larva ubiquitously expressing a hemopexin-interfering Mmp1 transgene, Mmp1DN Pro-pex.f1, with the tubP-GAL4 driver; these larvae also have normal dorsal trunks. (H) Larva expressing a hemopexin-interfering Mmp1 transgene, Mmp1DN E225A.f1, ubiquitously with the tubP-GAL4 driver; this panel is presented at higher magnification to show that each trunk has 1 or 2 pinched regions (arrow).

When induced in the tracheal system specifically with btl- phenotypes of the DN Pro-pex mutants strongly suggest that the GAL4 or throughout larvae ubiquitously with tubP-GAL4, DN ubiquitously expressed hemopexin domain interferes with the Pro-pex did not induce the taut tracheae and dorsal trunk breaks function of Mmp1 in basement membrane tissue invasion. As seen in the Mmp1 catalytic loss-of-function mutants (Table 1); with the Mmp1 hemopexin-deficient mutants, the disc eversion indeed these larvae appeared indistinguishable from wild-type phenotypes were strong but variable (Table 1). Ubiquitous (Fig. 2) and nearly all survived to pupariation, indicating that the overexpression phenotypes were examined in 2 lines carrying hemopexin domain of the dominant-negative protein does not independent insertions of DN Pro-pex and in a recombinant line interfere with larval Mmp1 functions in the tracheae. During containing both insertions; all caused widespread pupal lethality metamorphosis, however, the ubiquitous expression of DN Pro- and disc eversion failures, and yet none interfered with larval pex was lethal. These mutants arrested development with disc eversion defects, phenocopying the Q273* mutants (Fig. 3 and Table 1). Because the Q273* mutant discs are unable to invade ABEmbryos Larvae basement membrane during disc eversion (19), the similar

wt 2 Q273* W439* wt 2 Q273* W439* 250 197 A B C 148 125 96 83 64 Mmp1 wt Mmp1Q273*/W439* Mmp1Q273* 50

D E F 37 36 31

17 Mmp1W439* tubP>Mmp1DN pro-pex.f1 tubP>Mmp1DN E225A.f1 22 50 37 Fig. 3. Disc eversion phenotypes in pupae lacking functional Mmp1- hemopexin domains. Reflected light images of the anterior regions of pupae GAPDH 31 removed from their pupal cases are shown. (A) Wild-type at approximately 36 stage P5 with everted head (bracket) and wings (arrows); wings evert within 6 h after puparium formation (here they were pulled away from the body for Fig. 4. Western blots of Mmp1 mutants. (A) Each lane contains lysate better visualization) and the head everts Ϸ12 h after puparium formation. equivalent to 50 embryos of the following genotypes: w1118 wild type, null (B–F) Mmp1 mutants that have been allowed to develop several days after Mmp12 (control for antibody specificity), the hemopexin-deleted mutant puparium formation. (B) Mmp1Q273*/W439* transheterozygote with everted Mmp1Q273*, and the hemopexin-truncated mutant Mmp1W439*.(Lower) shows head and wings, not able to complete development. (C) Mmp1Q273* with GAPDH loading control. (B) Each lane contains lysate of second instar larvae of everted wings (arrows), but it has not everted its head and so appears headless the following genotypes: w1118 wild type, null Mmp12 (control for antibody (bracket). (D) Mmp1W439* that has not everted its head (bracket) or wings. (E) specificity), the hemopexin-deleted mutant Mmp1Q273*, and the hemopexin- Pupa expressing Mmp1DN Pro-pex.f1 ubiquitously with tubP-GAL4, with wing truncated mutant Mmp1W439*. The equivalent of 12 animals were loaded in all eversion (arrows) but no head eversion (bracket); black arrowhead shows the lanes except for Mmp12 with 18, because their small size required more larval mouth hooks, still attached to the body. (F) Pupa expressing animals to achieve equivalent loading. The highest molecular-weight band of Mmp1DN E225A.f1 ubiquitously with tubP-GAL4, that has everted wings (arrows) Mmp1, likely representing the uncleaved zymogen, is barely apparent in but not head (bracket). This genotype mimics the cryptocephalic phenotype of larvae in contrast to in embryos. (Lower) Loading control of GAPDH, which Mmp1Q273* but with higher penetrance. apparently is expressed differently in embryos and larvae.

2662 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804171106 Glasheen et al. Downloaded by guest on September 26, 2021 larvae, they also had increased tracheal breaks and appeared sluggish. In contrast, the range of disc eversion phenotypes observed from DN Pro-pex overexpression in the hemopexin- mutant background were similar to the Q273*/W439* back- ground (see Table 1), although the penetrance of head eversion failure was higher in the presence of DN Pro-pex. As larvae, these animals were still able to elongate their tracheae without breaks. We interpret these results to mean that the DN E225A mutant protein can interfere with the Mmp1 catalytic domain whereas DN Pro-pex interferes with Mmp1 via the hemopexin domain. From the null phenotype, it is clear that Mmp1 is required for tracheal remodeling during larval growth. To confirm the re- quirement for Mmp1 catalytic activity, rather than some unex- Fig. 5. Mmp1DN Pro-pex dominantly suppresses an Mmp1 overexpression pected function, we misexpressed the endogenous MMP inhib- phenotype. (A) Bristles on the thorax of a control fly appear long and tapered. itor Drosophila Timp. TIMPs inhibit the catalytic activity of Genotype: dpp-GAL4.(B) When Mmp1 is overexpressed, the bristles become MMPs with a 1:1 stoichiometry by occupying the active site (10), short, stubby and easily broken. Genotype: dpp-GAL4, UAS-Mmp1.f1, UAS- and fly Timp has been shown to inhibit Mmp1 both in vitro and GFP.(C) When Mmp1DN Pro-pex.f1 is coexpressed with Mmp1, the bristle phe- notype is significantly suppressed. Genotype: dpp-GAL4, UAS-Mmp1.f1, UAS- in vivo (18, 26). [Fly Timp can also inhibit Mmp2 (18, 26), but Mmp1DN Pro-pex.f1. there is no evidence that Mmp2 is required in the larval tracheal system (see Table 1).] Larvae expressing Timp throughout the tracheal system with btl-GAL4 displayed tightly stretched tra- development (Table 1). From the analysis of this dominant cheal tubes that developed breaks in their dorsal trunks at similar negative, and the hemopexin mutants, we conclude that the frequencies to those of Mmp1 null mutants (Table 1 and Fig. 2), hemopexin domain does not participate in larval tracheal re- confirming that the catalytic function of Mmp1 is required for modeling, whereas the hemopexin domain is required for Mmp1 normal elongation of the tracheal system during larval growth. function during disc eversion. The Mmp1 hemopexin domain of Thus, in Drosophila the Mmp1 catalytic domain is required Drosophila is specifically required during some functions but not during both larval tracheal elongation and during metamorphic others. tissue invasion. In contrast, the hemopexin domain is not required We tested another Mmp1 dominant-negative construct, DN for tracheal elongation but is required during the Mmp1-mediated E225A, which contains full-length catalytically dead Mmp1 with processes of tissue invasion during metamorphosis. a conserved active-site glutamic acid residue replaced with an Discussion alanine residue. The dominant-negative nature of this allele has been demonstrated in the embryonic nervous system and in cell We used the genetic strengths of Drosophila to examine the culture (24, 25). This mutant had the potential to interfere with function of an MMP hemopexin domain. We have defined the both the hemopexin and the catalytic domain. Interestingly, requirement for the hemopexin domain during metamorphosis BIOLOGY when expressed ubiquitously throughout the animal, larvae were by examining the phenotypes of Mmp1 hemopexin-disrupted DEVELOPMENTAL able to develop without tracheal breaks and survive to pupari- alleles Q273* and W439* and Mmp1 transgenes DN Pro-pex and ation; however, close examination of the larval tracheae revealed DN E225A. All of these mutant animals can remodel their small areas that appeared pinched (Fig. 2), suggesting that this tracheal tubes during larval growth but have severe defects in dominant-negative may interfere with the wild-type Mmp1 cat- metamorphosis consistent with failures in tissue invasion of alytic function. When expressed ubiquitously, the phenotype of basement membrane. Three independent genetic experiments show that the catalytic domain is required for tracheal elongation the DN E225A mutant was dramatic during metamorphosis: in larvae: the phenotype of Mmp12 null (P-excision) homozy- Pupae developed into cryptocephalic flies, with well-developed gotes, the phenotype of Mmp1Q112* null (EMS-generated) ho- bodies and wings, but without head eversion so that they mozygotes, and the phenotype of animals misexpressing the appeared headless. This phenotype was much less variable than MMP catalytic inhibitor Timp. Animals with any of these genetic the Mmp1 hemopexin allele phenotypes or the DN Pro-pex constitutions display stretched and broken tracheal tubes, indi- phenotype. cating that Mmp1-mediated proteolysis is required for normal To compare the nature of the 2 dominant-negative mutants, tracheal remodeling. Inhibition of Mmp1 catalysis during meta- we expressed each transgene in an Mmp1 mutant background. morphosis also causes disc eversion phenotypes, demonstrating We chose the Q273*/W439* background because stronger Mmp1 that the catalytic domain is required for both tracheal elongation alleles caused larval death even without dominant-negative and disc eversion (19). Mmp1 expression. We reasoned that, if DN Pro-pex protein is Four different genetic approaches to disrupting the he- out-competing Mmp1 for protein partners that bind to the mopexin domain function all show defects in pupal disc eversion, hemopexin domain, then the Q273*/W439* phenotype should be but the observed phenotypes are not uniform. Q273* homozy- mostly unchanged in the presence of DN Pro-pex, because this gotes, which lack the Mmp1 hemopexin domain entirely, have hemopexin-mutant Mmp1 never could bind to those protein phenotypes ranging from no eversion defects to severe defects, partners. Conversely, if DN E225A is competing with wild-type as do animals expressing the hemopexin-interfering DN Pro-pex Mmp1 for protein partners that bind both its hemopexin domain mutant. This phenotypic variability is also apparent in Mmp12 and its catalytic domain, then the Q273*/W439* phenotype null mutants whose larval defects have been partially suppressed should be greatly enhanced by the expression of DN E225A, by a mutation in Tubby (manuscript in preparation). This because the dominant-negative will interfere with the catalytic variability strongly suggests that there is partial genetic redun- function of the hemopexin-truncated proteins. As predicted, the dancy in disc eversion between Mmp1 and another gene. How- ubiquitous expression of DN E225A in the Q273*/W439* back- ever, the DN E225A phenotype is much tighter, with pupae ground caused severe defects in disc eversion, with most animals everting wings but failing to evert heads. This more uniform unable to evert either wings or head and stalling very early in phenotype suggests that DN E225A interferes with both Mmp1 metamorphosis (Table 1); this is a stronger phenotype than is and its partially redundant partner. Although the identity of the observed in either Q273*/W439* or tubϾDN E225A alone. As partially redundant partner is unknown, a good candidate is

Glasheen et al. PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2663 Downloaded by guest on September 26, 2021 Mmp2 because of its homology to Mmp1, because of the Mmp2 Materials and Methods head eversion phenotypes, and because DN E225A has been Drosophila Genetics. Drosophila melanogaster stocks used for this study are shown to interfere with both Mmp1 and Mmp2 in the embryonic listed in SI Materials. Mmp1 stocks were maintained over CyO, arm-GFP nervous system (24). balancers, and homozygous mutants were selected as late embryos or first instar larvae lacking GFP fluorescence. Larvae were maintained on yeasted Our data indicate that the Drosophila Mmp1 hemopexin agar plates until third instar, transferring them onto fresh plates every 1–2 domain participates selectively in some biological processes but days. Tracheal observations were made in live animals or heat-killed (by not others. How can this unexpected result be explained? It placing larvae on a coverslip and placing it on a 95°C block a few seconds until seems likely that the hemopexin domain is a protein–protein they stopped moving). interaction domain, as previously thought, but that it only binds to a subset of Mmp1’s binding partners and substrates; others Construction of Dominant-Negative Mmp1. For DN Pro-pex, mutagenic PCR was used to delete the region coding for the catalytic and hinge domains in both bind directly to the catalytic domain. These hemopexin binding splice forms Mmp1.f1 and Mmp1.f2 in pBluescript. The protein products of the partners could be substrates, localization determinants, or have resulting mutants were missing amino acids 112–298. The mutant ORF was other functions. It is reasonable to assume that Mmp1 substrates sequenced, ligated into pUAST and injected into Drosophila embryos. Similar in tracheal elongation and in disc eversion are different, because phenotypes were observed for the overexpression of DN Pro-pex.f1 and DN Pro-pex.f2. DN E225A.f2 was made according to (25), using the cDNA for the tracheae are lined with a chitinous apical extracellular matrix Mmp1 form 2. that must be remodeled during elongation, whereas imaginal disc eversion requires remodeling of a basement membrane contain- Western Blot Analysis. Sixty embryos 17–20 h AEL were homogenized in 36 ␮L ing IV and laminin (19, 27). Importantly, in addition to of 2X Laemmli Sample Buffer (LSB). A total of 30 ␮L of lysate, equivalent to 50 its role in developmental tissue invasion, Mmp1 is required in embryos, were loaded per lane. Twenty to fifty 2nd-instar larvae were ho- Drosophila for the local invasion of both transplanted and mogenized in 50 ␮Lof2ϫ LSB, and 12 larvae equivalents were loaded into each lane (18 for Mmp12 because of their small size). Proteins were transferred clonally induced tumors (19–21). These tissue invasion events to Hybond-C Extra (Amersham). Anti-Mmp1 monoclonals 3B8, 3A6, 5H7 were require the hemopexin domain, because it has been shown that used as 1:1:1 mixture diluted 1:10. These antibodies were all raised against the tumors induced in Q273* homozygous mutants have reduced catalytic domain of Mmp1 (18) and are available at the DSHB. Primary anti- invasive capacity; thus the hemopexin domain mediates both body incubation was overnight at 4 °C, and secondary antibodies (HRP- developmental and pathological tissue invasion (19). labeled goat-anti-mouse; Jackson) were diluted 1:5,000 and incubated for 1 h at RT. Loading was confirmed by goat anti-GAPDH (Imgenex) used at 1:5,000 Our data demonstrate that the domains of Drosophila Mmp1 for 2h at RT; followed by HRP-conjugated rabbit anti-goat (Jackson) used at do not contribute equally to the different developmental func- 1:10,000 for 1h at RT. Bands were detected using the Amersham ECL kit and tions for Mmp1. Rather, the hemopexin domain is dispensable film. for normal tracheal remodeling, whereas it is required for tissue invasion later at metamorphosis. These findings raise the pos- ACKNOWLEDGMENTS. We thank Gina Dailey for plasmid construction and Heather Broihier, Laura Lee, and Patrick Page-McCaw for comments on the sibility that a human MMP hemopexin domain may be a manuscript. The monoclonals used for this study are available at the Devel- candidate target for chemotherapies in patients. Thus, specificity opmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development. This work was of MMP inhibition could be achieved by targeting a subset of its supported by National Institutes of Health Grant R01GM073883 and by a Basil functions. O’Connor Starter Scholar’s Award from the March of Dimes.

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