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Transcriptional regulation of APH-1A and increased -secretase cleavage of APP and Notch by HIF-1 and hypoxia

Ruishan Wang^*,,1, Yun-wu Zhang^,1, Xian Zhang^*,, Runzhong Liu^*, Xue Zhang, Shuigen Hong^*, Kun Xia, Jiahui Xia, Zhuohua Zhang^,,2 and Huaxi Xu^*,,,2

^* Laboratory of Molecular and Cellular Neuroscience, School of Life Sciences, and Institute for Biomedical Research, Xiamen University, Xiamen, China;

National Laboratory of Medical Genetics of China, Xiang-Ya Hospital, Central South University, Changsha, China; and

Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla, California, USA

²Correspondence: Z.Z., National Laboratory of Medical Genetics of China, Xiang-Ya Hospital, Central South University, Changsha 410078, Hunan, China. E-mail: benzz{at}burnham.org; and H.X., Laboratory of Molecular and Cellular Neuroscience, School of Life Sciences, Xiamen University, Xiamen 361005, China. E-mail: xuh{at}burnham.org

SPECIFIC AIMS

The aims of this study are 1) to identify the promoter region of the APH-1A gene, which encodes a major component of the -secretase complex responsible for proteolytic cleavage of Alzheimer’s ßbeta;-amyloid precursor protein (APP) and the signaling receptor Notch; 2) to investigate transcription factors responsible for regulating APH-1A expression; and 3) to study whether transcriptional regulation of APH-1A gene affects its expression and thus the activity of -secretase for APP and Notch processing.

PRINCIPAL FINDINGS

1. Promoter analysis of the APH-1A gene
To determine the transcription initiation site of the human APH-1A gene, 5'-RACE was performed and the polymerase chain reaction (PCR) products were cloned into pGEM T vector for sequencing. The results indicated that the major transcription initiation site of the APH-1A gene is located 211 bp upstream of the translation start codon ATG (Fig. 1 A).

View larger version (30K): [in this window] [in a new window]   Figure 1. Sequence features and functional deletion analysis of the human APH-1A gene promoter. A) The 5'-flanking region of the human APH-1A gene was shown. The numbering was based on the transcription initiation site, which was grayed/boldfaced and defined as +1. The potential transcription factor binding sites were underlined/boldfaced. The protein-encoding sequence was italicized; m1 to m6 denote each transcription factor-binding site that was mutated for the mutagenesis study. B) Schematic diagram of the APH-1A promoter deletion constructs consisting of a 5' flanking region with serial deletions cloned into the promoterless vector pGL3-Enhancer. The numbers represent the end-points of each construct. The deletion plasmids were confirmed by sequencing and restriction enzyme digestion, and the digested samples were analyzed on a 1.5% agarose gel. C, D) The constructed plasmids were cotransfected with phRL-SV40 into HeLa cells (C) or rat cortical neurons (D). Luciferase activity was measured after 24 h with a luminometer. Renilla luciferase activity expressed by phRL-SV40 was used to normalize the transfection efficiency. The values represent means ± SE (n3), *P < 0.05 vs. pGL3-control, **P < 0.001 vs. pGL3-control (by ANOVA with the post hoc Newmann-Keuls test).

Furthermore, we PCR-amplified 15 fragments with different lengths of the 5' flanking region of the APH-1A gene and subcloned them individually into a promoterless luciferase plasmid pGL3-Enhancer (Fig. 1B ). These plasmids were transfected into HeLa cells (Fig. 1C ) and rat cortical neurons (Fig. 1D ). The promoter activity of different APH-1A fragments to drive luciferase expression was assayed. The results from HeLa cells and rat cortical neurons were similar (Fig. 1C, D ). The levels of luciferase gene expression driven by the longest APH-1A gene 5'-flanking region, the 926 bp fragment, was 20% of the positive control in HeLa cells and 26% in rat cortical neurons. Different deletions from both ends of the 926 bp fragment either increased or decreased luciferase activity, which suggests that both cis- and transacting regulatory regions exist between bp –746 to bp + 180. Luciferase activity driven by the 271 bp fragment was similar to the positive control, which suggests that the sequence from bp –233 to bp + 38 possesses a basic promoter apparatus.

2. The APH-1A gene promoter contains AP4 and HIF-1 binding sites
To identify potential transcription factor binding sites, we analyzed the sequence shown in Fig. 1A . Prediction analysis revealed that the core promoter region possesses two AP1 consensuses, three AP4 consensuses, and one HIF-1 consensus (Fig. 1A ). We mutated these potential binding sites individually and cloned them into the reporter vector. The promoter activity of these mutated fragments to drive luciferase expression was studied and compared with that of wild-type controls. Our results showed that mutations of the two AP1 sites (m1 and m2) had little effect on the promoter activity, whereas mutation of the HIF-1 site (m3) dramatically decreased the luciferase activity. Moreover, mutation of an AP4 site (m4) significantly reduced the luciferase activity, but mutations of the other two AP4 sites (m5 and m6) dramatically increased the luciferase activity. These results suggest that HIF-1 and AP4 are potential transcription factors regulating APH-1A expression.

3. AP4 and HIF-1 bind to APH-1A promoter in vitro
To investigate the binding of AP4 and HIF-1 to the promoter region, we performed EMSA. For AP4, we used the sequence corresponding to the APH-1A promoter region from bp –105 to bp –83 as a probe. Incubation of this probe with HeLa nuclear extracts resulted in a formation of a DNA-protein complex, which migrated at the same position of an AP4 consensus binding probe–protein complex (as positive control). The APH-1A AP4 probe–protein complex formation was dramatically inhibited by competitive binding with unlabeled AP4 consensus and APH-1A AP4 probe but not affected by competition from an SP1 consensus probe. These results indicate that the identified AP4 binding element can form a complex with nuclear AP4 protein in vitro.

Initial studies showed that incubation of the HIF-1 probes with HeLa nuclear extracts failed to produce any binding. However, when HeLa cells were treated with 1 mM NiCl₂ for 20 h (a condition of chemical hypoxia) and the nuclear extracts were incubated with the HIF-1 probes, a probe–protein complex was detected for both the probe corresponding to the APH-1A promoter region from bp + 100 to bp + 122 and a HIF-1 consensus probe. Competitive binding with unlabeled HIF-1 consensus and APH-1A HIF-1 probe significantly inhibited the complex formation, whereas competitive binding with mutated APH-1A HIF-1 probe and SP1 probe had little effect. These results indicate that the APH-1A HIF-1 binding element can form a complex with nuclear HIF-1 protein in vitro, when HIF-1 concentration is increased under NiCl₂ treatment.

4. HIF-1 regulates APH-1A expression
Due to a lack of any known reagents that can regulate AP4 activity, we focused our study on the roles of HIF-1 in APH-1A expression. It has been well known that HIF-1 activity is low under physiological conditions but increases dramatically under hypoxia. Some metals such as nickel and cobalt can increase HIF-1 activity and promote HIF-1-dependent transcription, mimicking hypoxic effects. Having shown that HIF-1 binds to APH-1A promoter region under NiCl₂ treatment in vitro, we further investigated whether HIF-1 regulates APH-1A expression in vivo. The APH-1A mRNA was significantly increased upon NiCl₂ treatments in a time-dependent pattern in HeLa cells. Concomitantly, the protein concentration of APH-1A was also dramatically increased upon 2 and 4 h NiCl₂ treatments, in response to increased HIF-1 concentration (Fig. 2 A).

View larger version (62K): [in this window] [in a new window]   Figure 2. Effects of HIF-1 activation on expression of -secretase components and proteolytic cleavage of APP and Notch. HeLa cells stably expressing human APP were treated with or without 1 mM NiCl2 for 2 or 4 h. A) Equal amounts of cell lysates were analyzed and immunoblotted with antibodies against HIF-1, APH-1A, PEN-2, PS1-NTF, nicastrin, APP/APP CTFs, and -tubulin, respectively. B) The conditioned media were assayed for sAPP by immunoblotting with antibody (Ab) 6E10 and for Aßbeta; by immunoprecipitation using Ab 4G8 followed by Western blot analysis using 6E10. For assaying Notch, cells were transiently transfected with NotchE construct and cell lysates were immunoblotted with anti-myc Ab 9E10 to detect Notch and NICD. The protein levels were quantified and normalized to controls. Note: For (A), the control was samples treated for 0 h; for (B), the control was the untreated samples at each time point. The ratios of NICD over Notch E were calculated and then normalized to controls. Data represent means ± SE from three separate experiments.

5. Chemical hypoxia increases -secretase mediated Aßbeta; and NICD formation
Although the APH-1 concentration was significantly increased upon short-term nickel treatment, the levels of APP, as well as the other three -secretase components, PEN-2, PS1, and nicastrin, remained unchanged. Interestingly, we found that Aßbeta; secretion was significantly increased upon 2 or 4 h treatment, accompanied with decreased APP CTFs and increased sAPP (Fig. 2B ), which indicates increased -secretase activity presumably as a result of increased APH-1A concentration. In addition, we found that short-term NiCl₂ treatments increased the steady-state levels of Notch NICD (Fig. 2B ).

CONCLUSIONS AND SIGNIFICANCE
The proteolytic cleavage of APP and Notch is mediated by the PS/-secretase complex, which consists of presenilins, nicastrin, APH-1, and PEN-2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. In the present study, we characterized the human APH-1A promoter region and demonstrated that AP4 and HIF-1 bind to the promoter. More importantly, we found that activation of HIF-1 by short-term NiCl₂ treatments (chemical hypoxia) dramatically increased APH-1A mRNA and protein expression. Although NiCl₂ treatments had little effect on APP and the other three -secretase components, we noticed an increased secretion of Aßbeta; accompanied by a decreased APP CTFs formation, indicative of an increase in -secretase activity. In addition, the secretion of sAPP, a derivative of APP through -secretase cleavage, was increased. Furthermore, the cellular concentration of Notch intracellular domain (NICD) was also increased by the chemical hypoxic treatment.

While no clear explanations for these perplexing results can be provided at present, we speculate the following: First, APH-1 may be the limiting factor for -secretase activity and increased APH-1 concentration alone may be sufficient to elevate the -secretase activity. Second, hypoxia has been known to affect membrane potential via mediating ion channels, hence nickel treatments or chemical hypoxia could change the intracellular/intramembranous microenvironments, rendering the substrates more susceptible to the -secretase. Finally, based on our recent findings that activation of protein kinase A (by forskolin) or MAPK by insulin/insulin-like growth factor-1 promotes secretion of both Aßbeta; and sAPP (or the trafficking of APP/Aßbeta;-containing vesicles from the trans-Golgi network), we could envision a similar stimulation by some yet unknown mechanisms involving HIF-1 activation. This may also possibly explain our observation of increased Notch cleavage. The formation of NICD has been thought to occur mainly at the plasma membrane, a site where -secretase was known to be highly active. A recent finding that hypoxia promotes the Rab11-mediated trafficking of the 6ßbeta;4 integrin, together with our previous finding that Rab11 is a key trafficking factor for the estrogen-stimulated APP secretion, provided indirect but reasonable support to the trafficking hypothesis.

Together, our results demonstrate that APH-1A expression and the -secretase mediated Aßbeta; and Notch NICD generation are regulated by hypoxia/HIF-1 (Fig. 3 ). The specific control of APH-1A expression by HIF-1 may imply physiological functions uniquely assigned to APH-1A.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schema of the features of the APH-1A promoter, and the hypoxia/HIF-1 regulated APH-1A expression and APP/Notch processing. The major transcription initiation site (TIS, numbered at +1) is at 211 bp upstream of the APH-1A protein coding sequence (CDS). Sequence prediction, mutagenesis, and gel shift assays showed that the APH-1A promoter possesses three AP4 binding sites and one HIF-1 binding site, which was predicted from bp + 103 to bp + 117. Nickel treatment (a condition of chemical hypoxia) or hypoxia increases HIF-1 concentration, which binds to HIF-1ßbeta; to form activated HIF-1 heterodimers. Activated HIF-1 promotes the transcription and translation of APH-1A. Increased APH-1A concentration probably promotes the -secretase activity, hence enhances the processing of APP to generate Aßbeta; and the cleavage of Notch to release NICD. Alternatively, it is possible that nickel/hypoxia changes the intracellular/intramembranous microenvironments or affects protein intracellular trafficking, rendering the substrates more susceptible to the -secretase. The dashed lines indicate hypothetical regulatory pathways.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5839fje

¹ These authors contributed equally to this work.

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5839fjev1 20/8/1275    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, R. Articles by Xu, H. Search for Related Content PubMed PubMed Citation Articles by Wang, R. Articles by Xu, H.