Figure 6

CystC and Δ14CystC inhibit NRK cell morphological transformation and anchorage-independent growth stimulated by TGF-β. (a) Normal rat kidney (NRK) fibroblasts were infected with control (namely pMSCV-IRES–GFP, where GFP stands for green fluorescent protein), CystC, or Δ14CystC retroviral supernatants as in Fig. 1a, and the resulting infected cells were isolated by GFP fluorescence on a MoFlo cell sorter 48 hours later. Shown in the upper panel are the GFP expression profiles of the resulting polyclonal populations. Also shown, in the lower panel, are recombinant CystC (CC) and Δ14CystC (Δ14) proteins present in conditioned medium of individual NRK populations and revealed by anti-CystC immunoblotting. (b) Control, CystC-expressing, and Δ14CystC-expressing NRK cells were cultured in soft agar in the absence or presence of transforming growth factor-β1 (TGF-β1; 5 ng/ml) for 7 days, whereupon NRK colony formation was quantified by light microscopy (upper panel). Middle panel, magnification of boxed regions. Lower panel, colony formation per microscope field (means ± SEM) observed in five independent experiments. (c) Control, CystC-expressing, or Δ14CystC-expressing NRK cells were transiently transfected with pSBE-luciferase and pCMV-β-Gal cDNAs, and were subsequently stimulated with TGF-β1 (5 ng/ml) for 24 hours. Afterward, luciferase and β-Gal activities contained in detergent-solubilized cell extracts were measured. Values are luciferase activities (means ± SEM) observed in three independent experiments normalized to maximal reporter gene expression induced by TGF-β in GFP-expressing cells. CystC expression significantly inhibited luciferase expression induced by TGF-β (*P < 0.05; Student's t-test). (d) Control, CystC-expressing, and Δ14CystC-expressing NRK cells were allowed to invade through Matrigel matrices in the absence or presence of TGF-β1 (5 ng/ml) for 48 hours. Values are means ± SEM for three independent experiments presented as the percentage invasion relative to GFP-expressing NRK cells. TGF-β1 significantly enhanced NRK cell invasion (*P < 0.05; Student's t-test). This TGF-β response was inhibited significantly by CystC and Δ14CystC expression (^#P < 0.05; Student's t-test), whereas tonic NRK cell invasion was significantly inhibited only by CystC expression (*P < 0.05; Student's t-test). TGF-β1 significantly enhanced NMuMG cell invasion (*P < 0.05; Student's t-test), a response that was inhibited significantly by CystC and Δ14CystC expression (^#P < 0.05; Student's t-test).