| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 36, 34286-34290, September 5, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Department of Biological Chemistry, Viale
G. Colombo 3, University of Padova and
¶Department of Histology, Microbiology, and
Medical Biotechnology, Via A. Gabelli 63, University of Padova, I-35121
Padova, Italy and the ||Department of Biochemistry,
Technical University of Braunschweig, Mascheroder Weg, 1, D-38124
Braunschweig, Germany
Received for publication, May 21, 2003 , and in revised form, June 16, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Several observations indeed point to a specific function of PHGPx in testis: (i) PHGPx activity in this tissue is higher than in any somatic tissue (17); (ii) expression of the corresponding gene, gpx-4, correlates with the abundance of maturating germ cells (18); (iii) expression level is under indirect control of gonadotropins (18); (iv) in mature spermatozoa it is found as an oxidatively inactivated protein that represents a major component of the mitochondrial capsule (16–19); (v) chimeric animals hemizygous for gpx-4 show mosaic testicular athrophy and abnormal sperm morphology (20) (vi) rescued enzymatic activity in clinical sperm samples correlates with fertility-related parameters (21); and (vii) promoter mutations in gpx-4 were detected in infertile subjects (22). More recently a selenoprotein band of 34 kDa, detected in testis (23), has been identified as a PHGPx variant with an N-terminal extension that comprises a nuclear targeting signal (24). This sperm nuclei, PHGPx ("sn GPx" in Ref. 24), is proposed to be involved in chromatin condensation and thereby to determine the differentiation of spermatogenic cells into spermatozoa (24).
How the nuclear PHGPx (nPHGPx) is formed is still debated. The PHGPx gene is composed of eight exons that, depending on species, are spread over 3 to 4 kb of DNA. It is transcribed into mRNAs of different lengths (25–27), which lead to three different isoforms differing in their N-terminal extensions: a cytosolic one (cPHGPx), a mitochondrial one (mPHGPx), and nPHGPx. cPHGPx and mPHGPx are derived from the same exon (designated 1A; see Fig. 1) by use of different translation starts, whereas the N-terminal extension of nPHGPx results from translation of an alternative exon (designated 1B; see Fig. 1). This phenomenon was first explained as resulting from alternative pre-mRNA splicing by Pfeifer et al. (24). The concept of splicing was also followed through by others (28). In the meantime, Moreno et al. (26) provided strong evidence for the existence of a second promoter that is located in the first intron and creates multiple transcription starts, including the alternative exon 1B, which is spliced to exons 2–7 and translated into nPHGPx. Attempts failed, however, to prove the activity of this alternative promoter region in reporter gene-transfected somatic cells (26, 27). This was interpreted by Borchert et al. (27) to support the original idea of alternative splicing of a transcript derived from the "joint basic promoter" upstream of exon 1A, whereas Moreno et al. (26), argue that the supposed alternative promoter is not recognized in somatic cells because it likely depends on transcription factors regulating nPHGPx expression specifically in germ cells.
|
|
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture—The rat thoracic aorta smooth muscle cell line A7r5 and the rat myoblast H9c2 cell line were grown in Dulbecco's modified Eagle's medium as described (29, 30) but supplemented with 50 nM sodium selenite.
Determination of the 5'-ends of gpx-4 Transcripts—The 5'-ends of PHGPx mRNAs were obtained by RACE cDNA amplification (SMART RACE cDNA amplification kit, Clontech) starting from total RNA of liver, heart, testis, and H9c2 cells. RNA was prepared using the RNeasy Mini-kit (Qiagen, Hilden, Germany) and retrotranscribed into 5'-RACE-ready cDNA samples of 1 µg each. The first 5'-RACE PCRs were performed with reverse primers targeting exon 1A (5'-caggccaggcacagccagag-3') or exon 1B (5'-caggaactcgtggctgttgctggtct-3') to gain the cytosolic and mitochondrial or nuclear transcripts, respectively. For the second amplification, nested reverse primers for exon 1A or 1B (5'-tgctggcttcaataagcggctcagac-3' or 5'-gttgaaaggctcgggaatgggct-3', respectively) were used. The agarose gel-purified RACE products were TA-cloned and sequenced.
Amplification of Rat Testis PHGPx mRNA—120 ng of retrotranscribed RNA was amplified (PCR master mix, Promega, Milano, Italy) in the presence of 8% dimethyl sulfoxide and 0.8 µm of the following primers for the mitochondrial transcript: reverse, 5'-gctgagaattcgtgcatgga-3', and forward, 5'-tccattggtcggctgcgt-3', or forward, 5'-agggcgggcaagccagac-3', or forward, 5'-attcaggcttcccattgg-3' or forward, 5'-ggcaacctcgcggataactg-3'); and for the nuclear transcript: reverse, 5'-atgcacacaagcccagga-3', and forward, 5'-ctgaacaaggccaggcct-3', or forward, 5'-tcggttagctagagtagg-3', or forward, 5'-tgggttactgggacctag-3'. The annealing temperature was 59 °C. Any genomic contamination of the retrotranscribed sample was ruled out by PCR with gpx-4-specific primers annealing to exons 1A and 4: forward, 5'-cctcgcgcgtccattggtcgg-3'; reverse, 5'-ccttgggctggactttcat-3').
Real-time PCR Measurements—Total RNA was again extracted using the RNeasy Mini-kit. Genomic contamination was ruled out as described above. Only samples with an A260/280 nm ratio of 1.8 were accepted.
Standard plasmids were prepared by TA cloning of PCR products obtained from rat testis cDNA with the following primers: forward, 5'-cctcgcgcgtccattgg-3', reverse, 5'-ccaggcaaaccatgtgc-3' (for the cytosolic/mitochondrial transcript); or forward, 5'-tctgctgcaggaccttccc-3, reverse, 5'-gctgagaattcgtgcatgga-3' (for the nuclear transcript). After propagation in Escherichia coli, plasmids containing the correct insert were purified (Plasmid Midi-kit, Qiagen) and quantified by A260 measurements.
Six hundred ng of RNA was retrotranscribed using the Taqman reverse transcription reagents kit (Applied Biosystems, Branchburg, NJ). Amplification was performed on an ABI PRISM 7007 sequence detection system (PE Applied Biosystems, Foster City, CA) and analyzed by Sybr green PCR core reagents (PE Applied Biosystems, Warrington, UK). The amplification mixture for nuclear transcript contained 3 mM MgCl2, 0.2 µM primers (forward, 5'-tctgctgcaggaccttccc-3'; reverse, 5'-gctgagaattcgtgcatgga-3') and serial dilutions of the retrotranscription mixture (1.5–15 ng of starting RNA). Standard reagents were adapted to a final volume of 25 µl. Amplification conditions were: 95 °C, 10 min; 95 °C, 15 s, and 63 °C, 1 min for 40 cycles. The amplification conditions of the cytosolic/mitochondrial transcripts were identical except for the concentration of MgCl2 (2 mM), the primers (0.4 µM; forward, 5'-ttattgaagccagcactgctgt-3'; reverse, 5'-gaattcgtgcatggagcgc-3'), and the annealing/extension temperature (65 °C).
Reporter Gene Experiments—Fragments of gpx-4 were generated by PCR with genomic DNA as template and gpx-4-specific primers and cloned into the KpnI/BglII site of the promoterless pGL3-Basic luciferase reporter vector (Promega). The primers were designed to yield the fragments –547 to –30, –547 to +422, –313 to +422, –179 to +422, +39 to +422, +86 to +422, and +86 to +649. The accuracy of the constructs was verified by sequencing.
Cells were seeded onto 24-well plates, grown to 70% confluence, and transfected with 0.2 µg of the above constructs and appropriate control vectors (pGLBasic vector for negative control and pRL-TK vector expressing Renilla luciferase (Promega) for control of transfection efficiency) using ExGen 500 (MBI-Fermentas, St. Lech-Rot, Germany). Cells were cotransfected with 0.2 µg of one of the above experimental constructs or 0.2 µg of empty vector. After a 24-h expression period, cell lysates were assayed for luciferase activity by the Dual-luciferase Reporter Assay System (Promega) on a plate-reading luminometer (Fluoroskan Ascent FL, Labsystem, Helsinki, Finland). Firefly luciferase activity of the experimental constructs was normalized by the Renilla luciferase activity obtained with the control vector pRL-TK and expressed as amplification factor in respect to the basic luminescence observed with pGLBasic/pRL-TK-transfected cells.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
A difference from the previously reported sequences of the N-terminal extension was observed. The sequence 12QCAGIRPYGP21 (24) is replaced here by 12CRQ14; this diversity was detected independently by others2 and thus is likely common. Otherwise, the total genomic sequence now established, –1497 to + 2714 (not shown; GenBankTM accession no. AJ537598 [GenBank] ), is remarkably congruent with published cDNAs (25) and partial genomic sequences (24). Just the insertion of a gctccgg motif upstream of the translation start of mPHGPx, if compared with the mRNA reported by Puspha-Rekka et al. (25), deserves to be mentioned.
An in silico analysis of the entire rat gpx-4 sequence from –1497 to + 2714 by the Genomatix PromoterInspector program (available at www.genomatix.de) suggested two distinct promoter areas (Fig. 1). The most upstream one corresponds in sequence and location to the promoter first identified by Huang et al. (35) in human GPX-4 and confirmed to be active by Maiorino et al. (22) and Borchert et al. (27). Being upstream of exon 1A, it may reasonably be presumed to regulate the products derived from this exon, i.e. cPHGPx and mPHGPx. The second promoter option is located in the first intron upstream of the alternative exon 1B. It is homologous to the mouse promoter postulated by Moreno et al. (26) to regulate the expression of nPHGPx, which depends on the transcription of exon 1B. This hypothetical promoter comprises 308 nucleotides that, according to MatInspector (36), contain 29 putative binding sites for transcription factors or regulatory proteins with a core similarity of 1 and an "optimized" matrix similarity. With identical criteria, a further 17 putative binding sites were identified in the region 340 nucleotides upstream of the putative promoter. In contrast to the corresponding region of the mouse gpx-4, no testis-specific regulatory elements were suggested in this area by the MatInspector program.
Typical splice sites that might yield an mRNA translating into nPHGPx, as suggested by Pfeifer et al. (24) and Borchert et al. (27), could not be detected irrespective of the assumed transcription start.
Proof of Second Promoter Activity—Left with no reasonable source to explain nPHGPx formation by alternative splicing and encouraged by the in silico prediction of the two promoters in gpx-4, we decided to reinvestigate the promoter activities with conventional luciferase-based reporter gene constructs and to test them in homologous, i.e. rat, mesenchymal cell lines.
Expectedly, the sequence –547 to –30 comprising the canonical upstream promoter only was active in both the embryonic rat heart myoblast line H9c2 and the smooth muscle cell line A7r5 (Fig. 2). If the whole stretch from –547 up to the translation start of exon 1B was lined to the reporter gene, activity declined to about 50% in both cell lines, suggesting that downstream elements bind inhibitory ligands. The removal of sequences upstream of the predicted promoter did not significantly affect the results. So far, the data is in line with the canonical promoter being localized in front of exon 1A. The constructs, starting at + 39 and + 86, however, which totally lack the canonical promoter, also showed significant activity in H9c2 cells, whereas in the A7r5 cells their activity was marginal. Furthermore, in both cell lines, increased activity was observed by including exon 1B almost completely into the construct lacking the canonical promoter (+86 to +649). These findings validate the prediction of the PromoterInspector program and, for the first time, unambiguously demonstrate that the hypothetical second promoter of gpx-4 is active in a cell type-dependent manner.
Detection of Exon 1B-derived Transcripts—As expected, the RACE technology applied to mRNA isolated from whole testis detected transcripts corresponding to both exons 1A and 1B (Table I). The 1B transcript extended 14 bases into the 5'-UTR, which agrees perfectly with the major transcription start region at –12 to –14 upstream of the AUG translation site of nPHGPx, worked out by Moreno et al. (26) for the mouse system. Surprisingly, however, an identical transcript could also be detected with mRNA isolated from heart and liver. This clearly indicates that the use of the alternate promoter is not restricted to germ line cells, and accordingly, nPHGPx is not a testis-specific protein.
|
In the H9c2 cells, which had been shown to use the alternate promoter efficiently, a similar and two additional RACE products were obtained, indicating additional transcription sites at –18, –73, and –108 relative to the nuclear translation start, which complies with the additional upstream transcription sites detected in the mouse system (26). Under our conditions, probing testis mRNA for the region upstream to exon 1A or 1B by reverse transcription PCR with suitable reverse primers and a series of forward primers nesting further 5' to the uppermost transcription start sites identified for each exon product by RACE, a corresponding series of the correct-sized bands was observed (Fig. 3). This suggests that, in both cases, transcription may be initiated even further upstream than suggested by RACE experiments.
|
In agreement with previous observations (25, 33), some of the exon 1A-derived transcripts identified here (Table I) do not cover the translational start of the mitochondrial PHGPx. This observation leaves open the possibility that the expression of gpx-4 into mPHGPx or cPHGPx, respectively, is also determined by alternative transcription and not only by an alternative use of the translation start site.
Quantitative Transcript Analysis by Real-time PCR—Transcripts encoding the exon 1A-derived products cPHGPx and mPHGPx and the 1B-derived nPHGPx were detectable by real-time PCR in somatic cell lines and all tissues investigated, i.e. testis as well as brain, heart, liver, and kidney. In each case nPHGPx transcript level were substantially lower than those encoding the cytosolic and mitochondrial forms (Table II). In terms of percentage of total PHGPx transcripts, the nPHGPx transcript varied between 17% in testis and 0.4% for the A7r5 cell line that had shown low activity of the alternative promoter region. The data reveals that nPHGPx is most abundantly expressed in testis in terms of both, absolute and relative amounts, but transcription of gpx-4 into nPHGPx-encoding mRNA is not restricted to spermatogenic cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The abundance of gpx-4 transcripts in testis underscores the relevance of the PHGPx to spermatogenesis and spermiogenesis, and indeed all of the three different forms may be equally important. Morphological alterations of spermatozoa, as were reported for hemizygous mice (20) and clinical sperm samples of infertile patients (21, 37), are best explained by the role of PHGPx as a structural protein of the mitochondrial capsule in sperm. Although only cPHGPx were detected in the capsule material (16), both mPHGPx and cPHGPx may be considered to provide the raw material for the capsule formation, because these two forms can no longer be distinguished after processing. However the oligospermia consistently observed in transgenic animals (20), in severely selenium-deficient experimental animals (38), and in infertile males with low PHGPx content in sperm (21, 37) points to a distinct role for the active enzyme in earlier stages of spermatogenesis, the PHGPx form involved being unclear. The appearance of nPHGPx in late spermiogenesis (24, 26, 28) coincides with DNA condensation. The ability of PHGPx to act on -SH groups of protamine as alternate substrates (24, 39) prompted the idea that its nuclear variant is indispensable for chromatin condensation by cross-linking protamine thiols. This process is also considered to guarantee appropriate sperm function and arrest of proliferation, once the germ line cells have reached their final state of differentiation (24, 26).
Under consideration for this postulated role of nPHGPx in sperm differentiation, its ubiquitous expression documented here may offer a key to understanding the essential nature of PHGPx in embryonic development. Surprisingly, all efforts to obtain homozygous PHGPx (–/–) mice have failed thus far.3 This outcome of gene disruption experiments cannot be explained by any of the established roles of PHGPx. Knock-out of gpx-1 and gpx-2 yielded offspring with a mild phenotype. GPx-1 (–/–) mice developed normally and displayed no phenotype whatsoever as long as they were not oxidatively challenged. Appropriate controls demonstrated that GPx-1 is the GPx variant that dominates antioxidant defense (40), revealing that a disturbed hydroperoxide metabolism cannot account for prenatal lethality. Similarly, the gastrointestinal GPx-2 could be knocked out without impairing development. Even when cross-bred with GPx-1 (–/–) mice, the resulting double knock-out mice were vital and only later developed an intestinal inflammatory syndrome (41). It is thus extremely unlikely that PHGPx, which is less abundant than any of its congeners, is of vital importance because of its antioxidant potential. Modulation of cytokine responses, as reported for gpx-4 overexpression, cannot likely be reconciled with embryogenesis either, because it has been similarly reported for GPx-1 (42), and needless to say, the role of PHGPx as a structural protein of spermatozoa is irrelevant to embryonic development. Rather a unique role of PHGPx in cellular differentiation would be compatible with the outcome of the reverse genetics approach. The use of protamine -SH groups as an alternate substrate of PHGPx, as demonstrated for testis (39, 24), might be taken as a model for a more general principle of how the enzyme might regulate cell division, and the nuclear form would surely be predestined to fulfill such demands.
|
FOOTNOTES |
|---|
* This work was supported by the MIUR (Ministero dell'Istruzione,
dell'Universitá e della Ricerca) Cofin 2002 (to F. U.) and Deutsche
Forschungsgemeinschaft Grant Fl 61/12-2 (to L. F.). The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 39-049-8276103; Fax:
39-049-8073310; E-mail:
matilde.maiorino{at}unipd.it.
1 The abbreviations used are: GPxs, glutathione peroxidases; cGPx, cellular
glutathione peroxidase; PHGPx, phospholipid-hydroperoxide glutathione
peroxidase; cPHGPx, cytosolic PHGPx; mPHGPx, mitochondrial PHGPx; nPHGPx,
nuclear PHGPx; RACE, rapid amplification of cDNA ends; UTR, untranslated
region.
2 R. Brigelius-Flohé, personal communication.
3 M. Brielmeyer, personal communication.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Ufer, C. C. Wang, M. Fahling, H. Schiebel, B. J. Thiele, E. E. Billett, H. Kuhn, and A. Borchert Translational regulation of glutathione peroxidase 4 expression through guanine-rich sequence-binding factor 1 is essential for embryonic brain development Genes & Dev., July 1, 2008; 22(13): 1838 - 1850. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Puglisi, A. Bevilacqua, G. Carlomagno, A. Lenzi, L. Gandini, M. Stefanini, F. Mangia, and C. Boitani Mice Overexpressing the Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase in Male Germ Cells Show Abnormal Spermatogenesis and Reduced Fertility Endocrinology, September 1, 2007; 148(9): 4302 - 4309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Walshe, M. M. Serewko-Auret, N. Teakle, S. Cameron, K. Minto, L. Smith, P. C. Burcham, T. Russell, G. Strutton, A. Griffin, et al. Inactivation of Glutathione Peroxidase Activity Contributes to UV-Induced Squamous Cell Carcinoma Formation Cancer Res., May 15, 2007; 67(10): 4751 - 4758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Mittapalli, J. J. Neal, and R. H. Shukle Antioxidant defense response in a galling insect PNAS, February 6, 2007; 104(6): 1889 - 1894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Imai, M. Saito, N. Kirai, J. Hasegawa, K. Konishi, H. Hattori, M. Nishimura, S. Naito, and Y. Nakagawa Identification of the Positive Regulatory and Distinct Core Regions of Promoters, and Transcriptional Regulation in Three Types of Mouse Phospholipid Hydroperoxide Glutathione Peroxidase J. Biochem., October 1, 2006; 140(4): 573 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Borchert, C. C. Wang, C. Ufer, H. Schiebel, N. E. Savaskan, and H. Kuhn The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis J. Biol. Chem., July 14, 2006; 281(28): 19655 - 19664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Maiorino, A. Roveri, L. Benazzi, V. Bosello, P. Mauri, S. Toppo, S. C. E. Tosatto, and F. Ursini Functional Interaction of Phospholipid Hydroperoxide Glutathione Peroxidase with Sperm Mitochondrion-associated Cysteine-rich Protein Discloses the Adjacent Cysteine Motif as a New Substrate of the Selenoperoxidase J. Biol. Chem., November 18, 2005; 280(46): 38395 - 38402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Conrad, S. G. Moreno, F. Sinowatz, F. Ursini, S. Kolle, A. Roveri, M. Brielmeier, W. Wurst, M. Maiorino, and G. W. Bornkamm The Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase Is a Protein Thiol Peroxidase Contributing to Sperm Chromatin Stability Mol. Cell. Biol., September 1, 2005; 25(17): 7637 - 7644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Banning, S. Deubel, D. Kluth, Z. Zhou, and R. Brigelius-Flohe The GI-GPx Gene Is a Target for Nrf2 Mol. Cell. Biol., June 15, 2005; 25(12): 4914 - 4923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bierl, B. Voetsch, R. C. Jin, D. E. Handy, and J. Loscalzo Determinants of Human Plasma Glutathione Peroxidase (GPx-3) Expression J. Biol. Chem., June 25, 2004; 279(26): 26839 - 26845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Jin, S. Baek, H. Lee, M. Y. Oh, Y. E. Koo, M. S. Shim, S. Y. Kwon, I. Jeon, S. Y. Park, K. Baek, et al. A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription Nucleic Acids Res., April 30, 2004; 32(8): 2482 - 2493. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
