Expression of cathepsin P mRNA, protein and activity in the rat choriocarcinoma cell line, Rcho-1, during giant cell transformation

Abstract

Lysosomal proteases perform critical functions in protein turnover and are essential for normal growth and development. Cathepsin P is a member of a newly discovered family of lysosomal cysteine proteases uniquely expressed in rodent placenta (PECs), and is closely related to human cathepsin L. Using the rat choriocarcinoma cell line model, Rcho-1, mRNA for the PECs cathepsins P, M, Q, R, 1, 2 was found to increase in expression during differentiation into a trophoblast giant cell phenotype. By contrast, expression of cathepsin L was not regulated. A specific enzyme assay was developed to show that activity of cathepsin P mirrored mRNA expression during differentiation. Cathepsin P protein co-localizes with cathepsin B, indicating that the enzyme probably functions in the endosomal-lysosomal compartment. This study demonstrates that the PEC genes produce functional proteases that can perform specific placental roles that are probably performed by broader specificity proteases in human placenta.

Figure 1. Cathepsin P expression is elevated in cells with a trophoblast giant cell phenotype

Phase contrast images of undifferentiated cells (panel A) and differentiated Rcho-1 cells (10 days, panel B) show morphological differences between the cells. In panel B, arrows point to the enlarged nucleus and arrow heads point to the highly vesicularized cytoplasm of the differentiated cells. Total RNA was isolated from Rcho-1 cells before (0 days) and after differentiation (days 2, 7 and 10) into giant cells. RT/PCR was performed to evaluate expression of cathepsin P and genes characteristic of TGs (PL-1, Hand1 and Stra13). β-Actin expression was examined as a loading control (panel C).

Figure 2. Expression of several PECs increase during Rcho-1 differentiation

Total RNA was isolated from Rcho-1 cells before (0 days) and after differentiation (days 4 and 7) into giant cells. RT/PCR was performed to evaluate expression of cathepsins L, M, P, Q, R, 1 and 2. Placental lactogen-I was analyzed as a marker of differentiation and β-Actin expression was examined as a loading control.

Figure 3. Real Time PCR quantitation of expression of cathepsins P and L during differentiation of trophoblast cells

Total RNA was extracted from undifferentiated cells and differentiated cells (days 1, 2, 4, 7 and 10). Ct values were obtained for cathepsins P and L and β-actin. Changes in expression of cathepsins P and L relative to β-actin are shown, normalized to expression levels in undifferentiated cells. Error bars indicate standard deviations (3 samples each) of a representative experiment that was repeated three times.

Figure 4. Activity of cathepsin P in cells and tissues

Recombinant cathepsin P was assayed using MOCAc-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH₂ in the presence or absence of E-64 (panel A). For comparison, cathepsin B and L activity were assayed using Cbz-Phe-Arg-N-methylcoumarin in the presence and absence of E-64 using pure enzymes (panel A). Activities are normalized to activity in the absence of inhibitor. Rcho-1 cells at different times after differentiation were homogenized in activity buffer and cathepsin P activity was determined in the absence (♦) and presence of E-64 (▲) and CtP-I (●) (panel B). Cathepsin P and B activities were measured in tissues from pregnant mice (panels C and D) and pregnant rats (panel E). In panels B, C, D and E, activities were calculated per mg protein and are normalized to tissues that exhibited maximal activity. Error bars indicate standard deviations and are derived from at least 3 separate samples.

Figure 5. Cellular localization of cathepsins B and P in Rcho-1 cells

Rcho-1 (A–C), JEG3 (D–F), and PC12 (G–I) cells were fixed, permeabilized, and incubated with rabbit anti-cathepsin P and sheep anti-cathepsin B specific antibodies. Species-specific fluorescent antibodies (Texas red mouse anti-rabbit IgG antibody and FITC donkey anti-sheep IgG antibody) were used to identify the primary IgGs. Cathepsin B is shown as green (A, D, and G) and cathepsin P as red (B, E, and H). Merged images are shown in C, F, and I, with nuclei stained blue with DAPI.