Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros (original) (raw)
Identification of TIMP-2, a ureteric protein that rescues metanephric mesenchymes from apoptosis. In tissue culture medium, isolated rat metanephric mesenchymes of E13 rats initiate apoptosis within the first 24 hours (6, 7) and involute over the following 24 hours. In contrast, treatment of mesenchymes with medium conditioned by UB cells induces visually obvious growth of the tissue and enhances [3H]thymidine incorporation (8, 14).
Twenty liters of medium conditioned by UB cells (600 mg of protein) was fractionated by heparin-Sepharose chromatography. Both [3H]thymidine incorporation and the visual survival of isolated mesenchymes identified 3 separated peaks of activity (Figure 1a). The most active of these peaks (eluting at 0.5 M NaCl) was further fractionated by anion exchange (Figure 1b), hydrophobic interaction (Figure 1c), and gel filtration chromatographs (Figure 1d). This yielded a single 23-kDa protein (Figure 2) that stimulated [3H]thymidine incorporation and rescued isolated metanephric mesenchymes from apoptosis (Figure 3).
Isolation of TIMP, a metanephric mesenchymal growth factor, from medium conditioned by UB cells. Medium conditioned by UB cells was fractionated by heparin-Sepharose chromatography (a). Three peaks of activity were found (100 μg of each fraction was assayed), and fractions 30–40 were pooled and further purified by a Mono Q anion exchanger (b). Active fractions (fractions 42–48; 10 μg of each fraction assayed) were pooled and further purified by phenyl-Sepharose hydrophobic interaction chromatography (c). Activity copurified with a single protein that we purified to homogeneity by Superdex-75 gel filtration (d). In c and d, 10% volume of each fraction was assayed for [3H]thymidine incorporation.
Isolation of TIMP-2. Silver stain of SDS-PAGE gel of medium conditioned by UB cells, and active fractions from the heparin-Sepharose, anion exchange (2 μg each), hydrophobic, and gel filtration chromatographies (5% volume each).
Rescue of isolated E13 metanephric mesenchymes from apoptosis by TIMP-2. Metanephric mesenchyme involutes (a) and is replaced by apoptotic bodies (c) when cultured for 48 hours in tissue culture medium (MEM with 10% FCS). In contrast, the addition of rTIMP-2 (2 μg/mL) rescues metanephric mesenchymal cells from apoptosis (b). Compared with mesenchymes incubated in basal medium, few apoptotic bodies are found when rTIMP-2 is included (d). Scale bars in a and b: 125 μm. Scale bars in c and d: 10 μm.
Mass spectroscopic analysis and sequencing of tryptic peptides identified the protein as TIMP-2, a protease inhibitor. We confirmed that TIMP-2 is able to rescue isolated metanephric mesenchymes from apoptosis by using 2 additional preparations of TIMP-2 (see Methods). In these experiments, the ED50 of rTIMP-2 was 2 nM (n = 5). Saturating concentrations of either rTIMP-2 (40 nM) or TIMP-2 isolated from UB cells stimulated [3H]thymidine incorporation 2- to 4-fold. The growth activity of rTIMP-2 was abolished by denaturing the protein. Rescue of mesenchymes from apoptosis was also stimulated by rTIMP-1 (not shown). These data demonstrate that TIMP proteins are able to rescue metanephric mesenchymes from apoptosis and are in agreement with prior demonstrations that TIMP proteins stimulate growth of a variety of cell lines (17, 25, 27–30).
To determine whether TIMP-2 stimulated mesenchymal growth as a result of its antiprotease activity, we examined the effect of ilomastat (GM6001; refs. 15–17), a matrix metalloproteinase inhibitor. First, using zymography, we found that ilomastat (at dosages as low as 0.04 μM) was active against proteases present in the metanephric mesenchyme, whereas a control compound, lacking antiprotease activity, permitted prominent gelatinolytic activity at 64 kDa, an activity previously reported in metanephric mesenchymes (31). However, despite its activity against metanephric metalloproteinases, ilomastat did not stimulate [3H]thymidine incorporation in isolated mesenchymes (8 individual doses ranging from 0.04 to 25 μM were tested; n = 6 independent assays). These data indicate that an inhibitor of many matrix metalloproteinases (15), including metanephric proteases, has no growth-promoting activity in metanephric mesenchyme. We conclude that while some of TIMP-2’s mesenchymal growth activity may be due to matrix metalloproteinase inhibition, other mechanisms of growth stimulation are likely to be operative.
TIMP-2 in embryonic kidney. Because UB cells are immortalized by SV40 T antigen and may express proteins not normally found in the native ureteric bud, we sought to confirm that TIMP-2 is expressed by the ureteric bud during its invasion of the metanephric mesenchyme. Using RT-PCR in dissected ureteric buds and mesenchymes, we found that both mesenchyme and ureteric bud synthesized TIMP-2 (not shown).
To localize matrix metalloproteinases inhibitor activity of TIMP-2 in the developing kidney, we performed reverse zymography using isolated E13 mesenchymes and ureteric buds (Figure 4a). The assay demonstrated that despite the synthesis of TIMP-2 in both the ureteric bud and the metanephric mesenchyme, all detectable TIMP-2 (and TIMP-1) activity was associated with the ureteric compartment when the kidney initially forms at E13. These data demonstrate that TIMP-2 is produced and likely sequestered by the ureteric bud in vivo.
Location of TIMP-2 in embryonic kidney. (a) Reverse zymography of isolated E13 mesenchymes and ureteric buds showing prominent localization of TIMP-2 antiprotease activity in the ureteric compartment. (b and c) Confocal microscopy of TIMP-2 immunofluorescence; a three-dimensional X-Y projection of serial cuts through the kidney. TIMP-2 immunoreactivity (green = fluorescein) is found in the condensed metanephric mesenchyme but not in mesenchymal cells that are more distant from the ureteric bud (arrows). Particularly prominent staining is found in the basement matrix surrounding branches of the ureteric bud (arrowheads), which is defined by binding of D. biflorus, a lectin specific for the ureteric bud (red = rhodamine). Scale bar in b: 140 μm. Scale bar in c: 100 μm.
We further localized TIMP-2 using immunocytochemistry in embryonic kidneys. Consistent with the reverse zymogram, the most intense staining was in the ureteric bud, particularly at branches and clefts (Figure 4, b and c; see Figure 9a). This is consistent with recently published immunolocalizations of TIMP-2 in the embryonic kidney (32). In the metanephric mesenchyme, staining was evident in cells that surround the tips of the ureteric bud, an area of intense proliferation (33) called the condensed mesenchyme. Mesenchymal cells more distal from the ureteric bud were negative (arrows in Figure 4, b and c). These results indicate that TIMP-2 may be a regulator of mesenchymal proliferation and ureteric branching in vivo. Because TIMP-1 can also rescue metanephric mesenchymes from apoptosis, it may have similar activities as TIMP-2, although the expression and distribution of TIMP-1 in the embryonic kidney is not yet established.
TIMP-2 localizes to the basement membrane of the ureteric bud. (a) Immuno-EM of E14 kidneys showing the localization of TIMP-2 to basement membranes of a cleft between 2 branches of the ureteric tree. (b) In contrast, little TIMP-2 is found at the tips of the ureteric bud, an area devoid of basement membrane. Note the curvature of the basal surface of the epithelia that define the tip of the ureteric bud. M, mesenchyme. UB, ureteric bud. Scale bars: 200 nm.
Secretion of TIMP-2 from UB cells is regulated by mesenchymal proteins that regulate development of the ureteric bud. Metanephric mesenchymal growth is synchronous with UB invasion, and, as noted above, proliferating condensed mesenchymes that are rich in TIMP-2 surround each tip of the invading ureteric bud. This suggests that TIMP-2’s mesenchymal growth activity may be a mechanism linking ureteric invasion and metanephric growth. To test this, we examined whether mesenchymal factors that control ureteric development could also induce the ureteric bud to secrete TIMP-2. UB cells (Figure 5a) and ureteric buds (not shown) show intracellular storage as well as constitutive secretion of TIMP-2 (Figure 5b). GDNF (34), FGF-7 (35), and FGF-1 (14) are produced by the metanephric mesenchyme, bind to receptors in the ureteric bud as well as in UB cells, and regulate ureteric growth and branching (2–5, 35–39). When monolayers of UB cells were challenged with each of these mesenchymal factors, GDNF and FGF-7 induced an immediate additional secretion of TIMP-2, with a significant effect occurring by the first sampling point of 1 minute (Figure 5c). In contrast, addition of FGF-1 had no effect. Both the basal and stimulated release of TIMP-2 were abolished at 4°C. These data suggest that metanephric mesenchymes can alter their own growth by releasing a secretory pool of TIMP-2 from the ureteric bud. Furthermore, since the secretogogues of TIMP-2 also regulate ureteric bud development, these data provide a mechanism by which development of these 2 tissues can be synchronized.
Secretion of TIMP-2 from UB cells is stimulated by mesenchymal proteins that regulate growth and branching of the ureteric bud. (a) UB cells contain immunoreactive TIMP-2. (b) Constitutive secretion of TIMP-2 from UB cells in culture. UB cells were incubated in serum-free MEM, and aliquots collected at the indicated times were assayed by reverse zymography for TIMP-2. (c) Secretion of TIMP-2 is stimulated by GDNF and FGF-7 (100 ng/mL; P < 0.02) but not by FGF-1 (100 ng/mL). At 4°C, both basal and stimulated secretion of TIMP-2 are abolished. Quantification of TIMP-2 secretion was derived from laser densitometry of reverse zymograms. The curves are averaged from 4 independent experiments and analyzed by ANOVA. Scale bar in a: 100 μm.
TIMP-2 maintains GDNF-expressing cells. Because secretion of TIMP-2 from UB cells is stimulated by proteins produced in the metanephric mesenchyme, such as GDNF and FGF-7, we examined whether mesenchymal cells expressing GDNF were among those rescued from apoptosis by TIMP-2. As shown in Figure 6, mesenchymes incubated with rTIMP-2 contained readily detectable GDNF, whereas it was only weakly detected in the absence of rTIMP-2, suggesting that TIMP-2 contributes to the viability of GDNF-producing cells. The results raise the possibility of a positive-feedback loop between the mesenchyme and ureteric bud that could help synchronize the development of the 2 tissues.
GDNF in metanephric mesenchymal cells that were rescued from apoptosis by incubation with TIMP-2. GDNF was detected by immunoblots in 6 freshly isolated E13 mesenchymes (0 hours) and in 6 mesenchymes maintained in culture by rTIMP-2 for 48 hours (48 hours + TIMP-2). In contrast, little reactivity remains in 6 untreated mesenchymes (48 hours). Standard is 25 ng of recombinant rat GDNF.
TIMP-2 contributes to ureteric bud morphogenesis. Epithelial morphogenesis in the developing breast, salivary gland, mandible, and kidney is regulated by the anti–matrix metalloproteinase activity of TIMP-1 (16, 31, 40–42). Moreover, our finding that mesenchymal factors that regulate ureteric development can induce secretion of TIMP-2 suggests that this protein may also be involved in ureteric bud morphogenesis.
To examine the role of TIMP-2 in the development of the ureteric bud, we incubated explanted E13 kidneys with rTIMP-2 and viewed the ureteric bud with the lectin D. biflorus. We found that kidneys cultured with rTIMP-2 (2 μg/mL) for 4 days had a 43% reduction in the number of ureteric bud tips compared with control kidneys (n = 23 for treated and control kidneys, P < 0.02; Figure 7, a and b). To determine whether inhibition of ureteric bud branching by TIMP-2 might be due to its anti–matrix metalloproteinase activity, we treated kidneys with the matrix metalloproteinase inhibitor ilomastat (15–17). Similar to the effect of TIMP-2, ilomastat (2 μM) inhibited branching by 53% when compared with embryonic kidneys treated with a control reagent for ilomastat, _N_-t-butyloxycarbonyl-L-leucyl-L-tryptophan methylamide, which had no effect (n = 35 for each group, P < 0.001; Figure 7c).
TIMP-2 inhibits ureteric branching in vitro. E13 kidneys were cultured for 4 days with rTIMP-2 (2 μg/mL), and the ureteric bud was then viewed with D. biflorus lectin. Treatment with rTIMP-2 inhibits branching of the ureteric bud (b), as compared with kidneys cultured in basal medium (a). Ilomastat (2 μM), an inhibitor of matrix metalloproteinases, also inhibited branching of the ureteric bud. Images are three-dimensional X-Y projections of serial confocal cuts through the kidney. Scale bars: 300 μm.
We also found that TIMP-2 may alter the composition of the basement membrane of the ureteric bud. Collagen IV immunoreactivity in the basement membrane of the ureteric bud was markedly enhanced in embryonic kidneys treated with rTIMP-2. Furthermore, in these kidneys collagen IV became detectable in the tips of the ureteric bud (Figure 8, a and b), a site normally devoid of basement membrane in vivo (43, 44). These effects of TIMP-2 are likely due to its inhibition of metanephric mesenchymal matrix metalloproteinases, because ilomastat also stimulated matrix accumulation (Figure 8c), even at the tips of the branches of the ureteric bud. The control reagent for ilomastat had no effect (not shown).
TIMP-2 enhances matrix deposition. E13 kidneys were cultured for 4 days with rTIMP-2 (2 μg/mL) and stained with antibodies to collagen IV. The entire ureteric tree is stained by collagen IV antibodies after rTIMP-2 treatment (b), including tip regions (arrows), while only proximal parts of the ureteric bud are obviously ensheathed by collagen IV in control kidneys (a). A few tips of the ureteric bud are indicated (arrow). (c) Similar to the effect of rTIMP-2, treatment of E13 kidneys with ilomastat (2 μM) resulted in matrix deposition throughout the ureteric tree. X-Y projection of serial cuts through the kidney. Scale bars: 100 μm.
Finally, by immuno-EM of the ureteric bud in vivo, we found few anti–TIMP-2 gold particles at the tips of the ureteric bud at the ureteric-mesenchymal interface, whereas along branches and in clefts that separate the branches of the bud, there were 2.4 ± 0.3– and 3.7 ± 0.9–fold (mean ± SEM) more anti–TIMP-2 gold particles per micrometer, respectively (P < 0.01; Figure 9). At these sites, TIMP-2 was abundant in the basement membrane of the ureteric bud. In contrast, at ureteric bud tips there was no basement membrane and little TIMP-2 (Figure 9 and refs. 43, 44).
Taken together, these data suggest that TIMP-2, once secreted from the ureteric bud, associates with its basement membrane where, by inhibiting matrix metalloproteinases, it increases deposition of collagen IV and perhaps other matrix constituents. This could inhibit further branching of the bud and would be consistent with the findings that TIMP can alter matrix composition in the mammary gland (45). Lastly, the data are concordant with the recent finding that GDNF, a secretogogue of TIMP-2, enhances matrix deposition by ureteric buds (46).