A novel function for hydroxyproline oxidase in apoptosis through generation of reactive oxygen species

Abstract

Proline and hydroxyproline are metabolized by distinct pathways. Proline is important for protein synthesis, as a source of glutamate, arginine, and tricarboxylic acid cycle intermediates, and for participating in a metabolic cycle that shuttles redox equivalents between mitochondria and cytosol. Hydroxyproline, in contrast, is not reutilized for protein synthesis. The first steps in the degradation of proline and hydroxyproline are catalyzed by proline oxidase (POX) and hydroxyproline oxidase (OH-POX), respectively. Because it is well documented that POX is induced by p53 and plays a role in apoptosis, we considered whether OH-POX also participates in the response to cytotoxic stress. In LoVo and RKO cells, which respond to adriamycin with a p53-mediated induction of POX and generation of reactive oxygen species, we found that adriamycin also induced OH-POX gene expression and markedly increased OH-POX catalytic activity, and this increase in activity was not observed in the cell lines HT29 and HCT15, which do not have a functional p53. We also observed an increase in reactive oxygen species generation and activation of caspase-9 with adriamycin in a hydroxyproline-dependent manner. Therefore, we hypothesize that OH-POX plays a role analogous to POX in growth regulation, ROS generation, and activation of the apoptotic cascade.

FIGURE 1.

Hydroxyproline and proline metabolism. Hydroxyproline and proline are metabolized, as shown above, by OH-POX and POX, respectively. P5CR, Δ¹-pyrroline-5-carboxylate reductase; P5CD, Δ¹-pyrroline-5-carboxylate dehydrogenase; TCA, tricarboxylic acid; KG, ketoglutarate.

FIGURE 2.

Adriamycin (Adr) induces OH-POX expression. RKO and LoVo cells were treated with and without adriamycin for 24 h. A, total RNA was prepared, and RT-PCR was performed for OH-POX and GAPDH as a control. The RT-PCR products were then run on an ethidium bromide gel. B, densitometry was performed on the bands from A, and levels of expression were normalized for GAPDH expression. The values represent means ± S.E. of the mean (n = 3). The control levels in LoVo cells were undetectable; however, both cell lines showed a significant increase (**, p < 0.01) in expression of OH-POX after induction with adriamycin.

FIGURE 3.

Adriamycin increases OH-POX catalytic activity. RKO, LoVo, HT 29, and HCT 15 cells were treated with and without 0.5 μm adriamycin. A, cells were collected at 24 h and lysed, and the activity of hydroxyproline oxidase was determined using an OAB spectrophotometric assay. The values represent means ± S.E. of the mean (n = 3). Adriamycin treatment significantly increased OH-POX enzymatic activity over control in RKO and LoVo cells (**, p < 0.01), which have wild type p53; however, there is no observable difference in OH-POX activity in the adriamycin-treated HT 29 or HCT 15 cells, which have mutant p53. B, RKO cells were treated as above, collected over time, and assayed for OH-POX catalytic activity. The values represent means ± S.E. of the mean (n = 3).

FIGURE 4.

Expression of p53 increases OH-POX enzymatic activity. RKO cells were transfected with equivalent amounts of pCi control vector (open circles) or p53 cDNA expression vector (closed circles). Cells were collected over time. Lysates were prepared, and protein was quantified and assayed for activity of OH-POX. The values represent means ± S.E. of the mean (n = 3). These data show that p53 alone is sufficient for inducing OH-POX catalytic activity, in the absence of adriamycin, and increases over time.

FIGURE 5.

Overexpression of p53 in cells with mutant p53 increases OH-POX enzymatic activity. The cell lines HT29 and HCT15 with mutant p53 were transfected with equivalent amounts of pCi control vector or p53 cDNA expression vector. After 6 h, the medium was replaced with new medium for 24 h. Cells were harvested, and cell lysates were quantified for protein and assayed for OH-POX activity. The values represent means ± S.E. of the mean (n = 3). By overexpression of p53, OH-POX enzymatic activity is significantly increased in both HT29 and HCT15 cells (**, p < 0.01).

FIGURE 6.

Hydroxyproline-dependent generation of ROS by adriamycin (Adr). A, RKO cells were grown in 6-well plates to 60–70% confluence and first transfected with siRNAs either for OH-POX or with OH-POX + POX and p53. The transfection medium was changed after 6 h and replaced with medium containing dialyzed serum, which has no hydroxyproline, and either 0 or 0.5 μm adriamycin (Adr) and/or 1 mm hydroxyproline (HYP). After 24 h, the cells were exposed to the fluorescent dye 2′7′-dichlorofluorescein diacetate. Fluorescence was normalized for protein content. The values represent means ± S.E. of the mean (n = 3). There is a significant hydroxyproline-mediated increase in ROS generation in the presence of hydroxyproline (**, p < 0.01) over cells treated with adriamycin alone. This effect is significantly reduced (++, p < 0.01) with the addition of siRNA for OH-POX. B, RKO cells were transfected with siRNA for OH-POX. After 24 h, cells were collected and assayed for OH-POX enzymatic activity. The values represent means ± S.E. of the mean (n = 3). Adriamycin treatment significantly increases (**, p < 0.01) OH-POX activity over control. However, treatment with siRNA for OH-POX significantly (++, p < 0.01) knocks down the increased activity of OH-POX in the presence of adriamycin.

FIGURE 7.

Hydroxyproline-dependent increase in caspase-9 activation. A, the RKO cells were treated with adriamycin (Adr) in the presence of various concentrations of hydroxyproline (HYP) as indicated for 24 h. Cell lysates were analyzed by Western blot analysis for activation of caspase-9. The graph shows the bands of activated caspase-9 analyzed by densitometry and normalized with actin. A representative result of several independent experiments has been shown. B, RKO cells were first transfected with siRNAs either for OH-POX or for OH-POX + POX. The transfection medium was changed after 6 h and replaced with medium containing dialyzed serum, which has no hydroxyproline, and either 0 or 0.5 μm adriamycin and/or 10 mm hydroxyproline. Cells were collected and lysed, and protein was quantified. Panel a, caspase-9 activity was determined colorimetrically. The results are cumulative of three independent experiments, and the values represent the means ± S.E. of the mean (n = 3). There is a significant hydroxyproline-mediated increase in caspase-9 activity in the presence of hydroxyproline (**, p < 0.01) over cells treated with adriamycin alone. This effect is significantly reduced (++, p < 0.01) with the addition of siRNA for OH-POX. A further decrease in activity was obtained with the addition of both siRNAs for OH-POX + POX as compared with the addition of siRNA for OH-POX (*, p < 0.05). Panel b, cell lysates were analyzed by Western blot analysis for cleavage of PARP. The bands of cleaved PARP were analyzed by densitometry and were normalized with actin. A representative result of several independent experiments has been shown.

FIGURE 8.

Proposed paradigm through which OH-POX can induce apoptosis. Under conditions of cytotoxic stress p53 mediates the up-regulation of both POX and OH-POX enzyme activities, catalyzing the conversion of proline to P5C and hydroxyproline to OH-3-P5C, respectively. During this process, electrons are generated to reduce oxygen resulting in the production of ROS, which can influence the mitochondrial membrane potential and be one of the mechanisms mediating apoptosis by releasing cytochrome c with the subsequent activation of caspase-9. P5CR, Δ¹-pyrroline-5-carboxylate reductase.