Chapter IV: An ECM protease/inhibitor network regulates cell outgrowth
4.4 Results
4.4.8 Effect of nematode astacins on C. elegans extracellular matrix
Meprins cleave extracellular matrix proteins (ECM) in vitro (Kohler et al., 2000; Kruse et al., 2004). Specifically, meprin α (Mep1A) cleaves laminin 1 on the α1 chain and cleaves laminin 5 on the α3 chain (Kohler et al., 2000), and both meprin α and meprin β (Mep1A and Mep1B) degrade collagen IV (Kruse et al, 2004), and proteolytically cut fibronectin and nidogen at several sites.
These studies indicate that meprin either degrades or cleaves components of the extracellular matrix so that metastasizing cells can move into surrounding tissues. We wanted to determine if the presence of meprin-like proteins (specifically NAS-21, NAS-22, and TOH-1) affect distribution of extracellular matrix proteins in uterine tissues. We tested this by observing changes in expression levels of ECM proteins through treating translation fusion expression constructs of ECM proteins with nas-21, nas-22, and toh-1 RNAi. We hypothesized that nas-21, nas-22, and toh-1 knockdown will increase protein levels of ECM components.
C. elegans collagen genes were ideal targets since both meprin subunits completely degrade collagen IV (Kruse et al, 2004). In order to determine if collagen was necessary for utse development, we performed RNAi against known C. elegans collagen genes (Table 1) and scored for utse defects. We chose these three genes specifically because two of these genes (emb-9 and let-
2) encode collagen IV in C. elegans, and mec-5, which is a collagen known to interact with protease inhibitor mec-1 (Emtage et al., 2004).
RNAi against mec-5 resulted in no utse defects (Table 1). Instead we focused on let-2 and emb-9.
RNAi against emb-9 generated utse defects (Table 1) and we used this protein to characterize the effect nematode astacins have on collagen distribution.
Changes in expression pattern were difficult to visualize using wide-field epifluorescence microscopy. We therefore used confocal imaging to finely observe changes in ECM expression due to nas-21, nas-22, and toh-1 RNAi.
We used a emb-9p::emb-9::dendra line to observe changes in emb-9 expression (Ihara et al., 2011).
No changes in emb-9 expression were observed in the presence of nas-21 and nas-22(RNAi) (Figure 8B and 8C); however, toh-1 RNAi did increase expression levels of emb-9 in the vicinity of the uterus (Figure 8D). emb-9 expression can be categorized into two types: puncta/globule-like accumulations of expression in the body wall, and a thin line of expression in the utse (Figure 8A).
In toh-1(RNAi) treated worms, we saw a large increase in the number of emb-9 globules in the body wall as well as an accumulation of gfp in the posterior uterine region (see red box Figure 8D). We therefore believe that toh-1 specifically regulates levels of emb-9 in the C. elegans uterus.
Since meprin α (Mep1A) cleaves laminin (Kohler et al., 2000), we also wanted to determine the effect of nas-21, nas-22, and toh-1 knockdown on C. elegans laminins. C. elegans has four laminins: two encode the laminin α chain (epi-1 and lam-3), one encodes the laminin β chain, epi- 1, and one encodes the laminin γ chain, lam-2 (Kramer 2005). We wanted to determine if these laminins involved in utse development, and to this end, we performed RNAi against these four genes and screened for utse defects (Table 1). Two of these genes, lam-1 and epi-1, show significant utse defects.
Though RNAi against epi-1 generates severe (Figure S7B-D) defects in utse, we saw that epi-1 is not expressed in the vicinity of the uterus (Figure S7E). Therefore, we eliminated epi-1 among the laminins potentially being regulated by nas-21, nas-22, and toh-1.
We used the lam-1 translational fusion, lam-1p::lam-1::gfp, to observe lam-1 expression (Ziel et al., 2009). lam-1 is expressed in the dorsal utse edge as well at the lumen of the C. elegans uterus and
in the distal tip cell (DTC) (Figure 8E). RNAi against nas-22 and toh-1 had no effect on lam-1 (Figure 8G and 8H). However, nas-21(RNAi) treated worms showed an increase in the intensity of lam-1 expression in the uterus and in the distal tip cells (Figure 8F). We wished to quantify the change in intensity of lam-1 expression so that we could determine the fold by which LAM-1 levels had increased. We chose two slices that were at comparable locations in the worm (based on vulval anatomy) from the lam-1p::lam-1::gfp and nas-21(RNAi) lam-1p::lam-1::gfp z-stacks (Figure 8M and Figure 8O). For each image, we quantified mean intensity in an area that contained the uterus (see whites boxes, Figure 8N and Figure 8P). Treatment of nas-21(RNAi) caused an 1.76 fold increase in fluorescent intensity. Therefore, we believe that presence of nas-21 is necessary to control levels of lam-1 expression (Figure 8A).
Since nas-21 is affecting levels of LAM-1, we wanted to see if knockdown protease inhibitors acting on nas-21 (cpi-1, srp-2, mec-1, F35B12.4) would affect levels of LAM-1. Ideally knockdown of protease inhibitors would increase nas-21 activity, which would increase amount of laminin cleavage by NAS-21 and reduce levels of lam-1 expression. srp-2, F35B12.4, and mec-1 RNAi treatment of lam-1p::lam-1::gfp did not affect lam-1 expression levels (Figure S8B-D). Since there are multiple genes that affect activity of nas-21, we believe that reducing the levels of these individual protease inhibitors by RNAi does not increase NAS-21 activity to the level where observable changes in lam-1 expression are apparent.
Nidogen and fibronectin are also cleaved or degraded by meprins (Kruse et al., 2004). nid-1 encodes the sole C. elegans nidogen homolog (Kang and Kramer 2000; Kim and Wadsworth 2000).
RNAi against nid-1 generated significant utse defects (Table 1). Antibody staining shows nid-1 staining within the dorsal edge of the uterus (Kang and Kramer, 2000), indicating that it may be acting in this region. However, nid-1::gfp expression is limited to the PLM neurons, the intestinal cells, and the distal tip cells of the gonad (Kim and Wadsworth 2000). Therefore we were unable to evaluate changes in uterine expression of nid-1 due to actacin knockdown.
C. elegans lacks fibronectin (Meighan et al., 2004); however, the C. elegans genome contains 23 genes that contain fibronectin domains (Table 1) We saw that RNAi treatment against two of these genes, unc-73 and unc-40, resulted in significant utse defects (Table 1). Interestingly, both of these genes are involved in branch formation (Struckhoff and Lundquist 2003; Hao et al., 2010), a phenotype we observed as a result of nas-21(RNAi) treatment and in nas-21(gk375710) (Figure 3C,
3D, 3G). We hypothesize nas-21 regulates both branch formation activity and fibronectin production by acting upstream of these genes.
We also wanted to use the utse to identify other meprin targets. One such target was the ECM protein sdn-1. sdn-1 is the C. elegans homolog of vertebrate syndecan, a type I transmembrane heparan sulfate proteoglycan (Kinnunen 2014). sdn-1 is expressed in the hypodermis, and is involved in HSN neuron migration. We used a sdn-1p::sdn::gfp reporter to visualize sdn-1 localization, and saw expression in the hypodermis in the vicinity of the uterus (Figure 8I). nas-21, nas-22, and toh-1(RNAi) treatment of sdn-1p::sdn::gfp resulted in an expansion of the sdn-1 expression pattern along the anterior posterior axis (Figure 8J-L), indicating that these three astacins may be cleaving/degrading sdn-1. Therefore, using our model we have identified an additional ECM protein that meprin-like nas-21, nas-22, and toh-1 act on (Figure 11).