Elsevier

Human Pathology

Volume 42, Issue 1, January 2011, Pages 75-87
Human Pathology

Original contribution
PINK1 displays tissue-specific subcellular location and regulates apoptosis and cell growth in breast cancer cells

https://doi.org/10.1016/j.humpath.2010.05.016Get rights and content

Summary

The PINK1 gene is mutated in the germ line of patients with hereditary early-onset Parkinson disease, and PINK1 prosurvival function at neuronal mitochondria has been related with the etiology of this disease. However, the expression and function of PINK1 protein in nonneuronal tissues has not been determined yet. Here, we have analyzed PINK1 protein expression and subcellular distribution in normal and neoplastic human tissues and investigated the function of PINK1 in breast carcinoma cells. PINK1 protein, as stained by a specific anti-PINK1 monoclonal antibody, was widely expressed in human tissues, displaying high expression in epithelial tissues and in the central nervous system and lower expression in tissues of mesenchymal origin. The subcellular distribution of PINK1 was cytoplasmic granular or cytoplasmic diffuse in most tissues. In breast, PINK1 was also associated with the plasma membrane. Human neoplastic tissues ranged from high PINK1 expression in carcinomas to low expression in sarcomas. In neoplastic tissues, PINK1 displayed a diffuse cytoplasmic localization, with an additional membranous localization in breast carcinoma and squamous carcinoma of lung. In the human breast carcinoma Michigan Cancer Foundation-7 cell line, ectopic expression of cytoplasmic or mitochondrial-targeted PINK1 inhibited apoptosis triggered by hydrogen peroxide and suppressed cell growth in soft agar, whereas PINK1 silencing increased hydrogen peroxide-induced apoptosis. Together, our findings indicate that the physiologic functions of PINK1 go beyond its regulatory role of mitochondria-mediated cell survival in neurons.

Introduction

Parkinson disease (PD) is the second most common neurodegenerative disease associated with movement disorders. It is linked pathologically to death of dopaminergic neurons projecting from the substantia nigra pars compacta to the striatum, and oxidative stress and mitochondrial dysfunction appear to play prominent roles in this cell death. Although most PD cases seem to be sporadic in nature, an estimated 10% of cases are familial and show hereditary genetic defects. The identification of these genes and the functions of their protein products are providing important insights into the molecular pathogenesis of PD [1], [2]. Germ line mutations in the PINK1 gene, which encodes the PINK1 protein, are linked to the Parkinson Disease 6-associated autosomal recessive form of Parkinsonism [3]. PINK1 messenger RNA (mRNA) is detected in several tissues [4], although the regulation of PINK1 transcription is mostly unknown. In this regard, the mRNA levels of PINK1 have been found to be overexpressed in different cancers with high metastatic potential as well as in cells forced to express ectopically the Phosphatase and tensin homolog deleted in chromose ten tumor suppressor [4], [5], making possible the involvement of PINK1 in the unbalanced apoptotic or metastatic processes of cancer.

PINK1 complementary DNA (cDNA) encodes a 581-amino acid protein of 63 kd, composed of a mitochondrial targeting sequence (amino acids 1-77), a putative transmembrane domain (amino acids 101-107), a serine/threonine protein kinase catalytic domain (amino acids 150-510), and a putative regulatory C-terminal tail (amino acid residues 510-581) [6], [7]. Once inserted into mitochondria, the mitochondrial targeting sequence is cleaved, generating a PINK1-processed protein of 55 kd. PINK1 is localized in the mitochondria, although the precise topology and localization of PINK1 in this organelle remains controversial [8], [9]. Several studies have demonstrated that PINK1 function is related to the inhibition of mitochondria-dependent apoptosis. Moreover, an artificial cytoplasmic PINK1 mutant has also been reported to be antiapoptotic in neurons [6], [10].

Using anti-PINK1 polyclonal antibodies, Gandhi et al [11] have shown the expression of PINK1 protein in normal and PD human brain. However, the distribution of endogenous PINK1 protein in brain and other peripheral tissues has not been established using monospecific anti-PINK1, and currently available anti-PINK1 antibodies are reported to exhibit poor specificity and/or sensitivity [9]. To study the expression of PINK1 protein in normal and disease-associated tissues, we have generated and characterized an anti-PINK1 monoclonal antibody (mAb) (called 89B mAb). We report here the expression pattern of PINK1 protein in normal and neoplastic tissues. Our results show that PINK1 protein expression is not restricted to the brain but rather displays a wide and heterogeneous tissue distribution, with tissue- and tumor-specific differential subcellular localizations. Furthermore, functional analysis of Michigan Cancer Foundation-7 breast carcinoma cells unveiled antiapoptotic and growth regulatory properties of PINK1 in this cell type. Our findings suggest that altered function and/or subcellular localization of PINK1 may play a role in oncogenesis.

Section snippets

Plasmid construction, purification of recombinant proteins, and polymerase chain reaction

The cDNA encoding full-length human PINK1 was obtained from Geneservice (Mammalian Gene Collection, IMAGE ID 5214483; GeneService, Cambridge, United Kingdom). As this DNA contains the mutation P209A, the original cDNA clone was changed by site-directed mutagenesis to reconstitute the wild-type Pro209. pRK5 PINK1-HA, pRK5 PINK1-HA 150-510, and pRK5 PINK1-HA 150-581 mammalian expression vectors (hemagglutinin-tagged C-terminal) were made by polymerase chain reaction (PCR) amplification of PINK1

Specificity of anti-PINK1 89B mAb

To study the expression of endogenous PINK1 protein in human tissues, we immunized mice with purified recombinant GST-PINK1 150-510 (PINK1 catalytic domain; human sequence) and generated a hybridoma clone (89B) secreting an anti-PINK1 IgG1 mAb. The 89B mAb reacted by enzyme-linked immunosorbent assay with GST-PINK1 150-510 but not with GST alone (data not shown). Moreover, the 89B mAb reacted by immunoblot with both GST-PINK1 150-510 purified from bacteria (data not shown) and PINK1-HA

Discussion

Our results provide the first description of PINK1 protein expression in normal and neoplastic human tissues. Several relevant observations arise from our study that can be important to understand the roles of PINK1 in human disease. First, we have found that PINK1 protein expression is not restricted to the brain but rather shows a wide tissue distribution. In particular, PINK1 protein was detected in epithelial glandular tissues, such as prostate or adrenal and mammary glands. This

Acknowledgments

The authors thank Robert P. C. Shiu for providing MCF-7 Tet-On cells. J. J-S. is the recipient of a fellowship from FIS-ISCIII-Ministerio de Sanidad (Spain).

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    This work was partially supported by grant SAF2006-083139 from Ministerio de Educación y Ciencia; grant SAF2009-10226 from Ministerio de Ciencia e Innovación; grants ISCIII-RETIC RD06/0020/0049 and RD06/0020/0102 from Ministerio de Ciencia e Innovación e Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional; grant ACOMP/2009/363 and ACOMP/2010/222 from Generalitat Valenciana (Spain and European Union); and a grant from Fundació Gent per Gent (Spain).

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    A. Berthier and S. Navarro contributed equally to this work.

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