Regulation of the expression of aminopeptidase A, aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators

Abstract

Aminopeptidase (AP) A is a transmembrane type II molecule widely distributed in mammalian tissues. Since APA expression may be absent in renal cell carcinoma (RCC), it is possible that there is an altered regulation or other defect of APA upon malignant transformation of proximal tubular cells. However, investigations into the regulation of APA on tumour cells are rare. We report, for the first time, that both transforming growth factor-beta 1 (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) down-regulate APA mRNA as well as protein expression in renal tubular epithelial cells and RCC cells in culture. In addition to this, both cytokines decrease dipeptidylpeptidase (DP) IV/CD26 mRNA, but not APN/CD13 mRNA expression. Otherwise, IL-4 and IL-13 increase CD13 as well as CD26 expression, but do not alter APA expression. Interferon-alpha (IFN-alpha), IFN-beta and IFN-gamma increase mRNA expression of all the three membrane ectopeptidases, whereas IL-1, IL-6, IL-7, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any significant effect. Treatment of cultured cells with cAMP-increasing agents, such as 8-bromo-cAMP or A23187, results in an increase in APA and DPIV/CD26, but no change in APN/CD13 mRNA expression or even a decrease in it. Furthermore, AP inhibitors can influence APA mRNA expression, since bestatin causes an increase in APA expression in a time- and dose-dependent manner, whereas bestatin does not change CD13 or CD26 expression. No difference could be found with respect to the modulation by different mediators between RCC cells and renal epithelial cells, though permanent tumour cell lines such as Caki-1 and Caki-2 may have lost some of the normally expressed peptidases.

Fig. 1

Comparison of the absolute amounts of the mRNA of aminopeptidase (AP) A, APN/CD13 and dipeptidylpeptidase (DP) IV/CD26 in renal tubular epithelial cells (n = 11) and renal cell carcinoma (RCC) cells (n = 10) in culture. Results of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) after scanning and calculation as described in Materials and Methods. Data are given as box plots with the median. The box encompasses the 25th to 75th percentiles. The 5th and 95th percentiles are displayed as error bars.

Fig. 2

Regulation of the mRNA expression of aminopeptidase (AP) A (□), APN/CD13 (▪) and dipeptidylpeptidase (DP) IV/CD26 () by different cytokines. Renal cell carcinoma (RCC) cells or renal epithelial cells were cultured for 24 h with: (i) IL-4 (200 U/ml); (ii) IL-13 (10 ng/ml); (iii) tumour necrosis factor-alpha (TNF-α) (100 U/ml); (iv) transforming growth factor-beta 1 (TGF-β1) (10 ng/ml); (v) IFN-α2b (1000 U/ml); (vi) IFN-β (1000 U/ml); (vii) IFN-γ (200 U/ml). Results of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) are given as a percentage of the basal control (culture without cytokine = 0%, n = 6). Statistically significant differences from control: *P < 0.05; **P < 0.01.

Fig. 3

Effect of different concentrations of bestatin on aminopeptidase (AP) A mRNA expression of renal cell carcinoma (RCC) cells and renal epithelial cells after a 24-h incubation by use of competitive reverse transcriptase-polymerase chain reaction (RT-PCR). On the ordinate the relative APA mRNA expression is given as a percentage of the control (culture without bestatin = 0%, n = 5).

Fig. 4

Effects of different mediators on mRNA expression of aminopeptidase (AP) A (□), APN/CD13 (▪) and dipeptidylpeptidase (DP) IV/CD26 (). Renal tubular epithelial cells or renal cell carcinoma (RCC) cells were cultured with A23187 (200 ng/ml), 8-bromoadenosine 3′5′monophosphate (100 μ m), forskolin (10 μ m), cholera toxin (1 μg/ml), or prostaglandin E₂ (PGE₂) (1 μ m) for 24 h. Results of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) are shown as a percentage of the control (culture without mediator = 0%, n = 6). Statistically significant differences from control: *P < 0.05; **P < 0.01.