Snail interacts with hPLSCR1 promoter and down regulates its expression in IMR-32

doi:10.1016/j.bbrc.2014.05.094

Biochemical and Biophysical Research Communications

Volume 450, Issue 1, 18 July 2014, Pages 172-177

https://doi.org/10.1016/j.bbrc.2014.05.094 Get rights and content

Highlights

•
Putative TFs binding sites for AML-1, Sox-5 and Snail within hPLSCR1 promoter.
•
⁻¹¹¹⁸CAGGTG⁻¹¹²³ is the Snail binding site in hPLSCR1 promoter.
•
Interaction of Snail with endogenous hPLSCR1 promoter confirmed by ChIP assay.

Abstract

Human phospholipid scramblase 1 (hPLSCR1) is a proapoptotic protein whose expression is deregulated in a variety of cancers cells. However till date the transcription regulation of hPLSCR1 is unknown. Transcriptional regulation of hPLSCR1 was studied by cloning the 5′-flanking region of hPLSCR1. Luciferase assays revealed that −1525 to −1244 region of hPLSCR1 was found to regulate its promoter activity. A putative Snail transcription factor (TF) binding site was found within the regulatory region of the promoter. Snail binding was found to down regulate the expression of hPLSCR1 both at the transcriptional and translational levels. Snail knock down using Snail-shRNA confirmed that down regulation of hPLSCR1 by Snail was specific. Point mutation studies confirm that the predicted Snail TF binds to −1123 to −1117 site. ChIP assay further confirms the physical interaction of Snail with hPLSCR1 promoter. This is the first report showing the transcriptional regulation of hPLSCR1 expression by Snail TF and its possible implications in cancer progression.

Introduction

Epithelial-mesenchymal transcription (EMT) is a crucial process required for the polarization of epithelial cells to invasive mesenchymal phenotype [1]. Important event during EMT transition involves down-regulation of cell adhesion molecule E-cadherin and till date several repressors of E-cadherin have been identified and termed as EMT regulators (EMTRs). EMTRs include Snail, Slug, ZEB1, SIP1 and Twist which are important for transition of epithelial cells to melanoma cells and also contribute to resistance towards apoptosis, senescence and immune system evasion [2], [3]. Snail and Slug which belong to the E-box class of transcription factors are the most important transcription factors which regulate E-cadherin expression [4]. Initially Snail was implicated in the differentiation of epithelial cells to mesenchymal cells during embryo development; however the same molecule has been shown to be crucial for tumor progression. Snail transcription factor is an important zinc finger protein which effects EMT transition via down regulation of E-cadherin and up-regulation of vimentin [5]. Expression of Snail is up-regulated in several cancers such as colorectal cancer, renal cell carcinoma and myeloid cancer underlying the importance of this molecule in tumor invasion and metastasis.

hPLSCR1 is a type II integral membrane protein belonging to the ATP-independent class of phospholipid translocators which mediate bidirectional scrambling of phospholipids (PLs) in a Ca²⁺ dependent manner. It is a multi-domain protein which effects bidirectional scrambling of PLs particularly phosphatidylserine (PS) which acts as a signal for apoptosis, blood coagulation and cell activation events [6]. In hPLSCR1−/− null mice, blood coagulation was normal however defective proliferation and differentiation of hematopoietic cells was observed [7]. Hence hPLSCR1 is not considered as a true scramblase despite mediating transbilayer movement of PLs in synthetic vesicles [8]. hPLSCR1 is known to shuttle between nucleus and PM via nuclear import pathway by importin α/β proteins [9]. In the nucleus hPLSCR1 binds to the inositol 1,4,5 triphosphate receptor type 1 (IP3R1) and up regulates its expression [10]. In addition, hPLSCR1 also interacts with topoisomerase and enhances the decatenation activity [11]. hPLSCR1 has been shown to regulate the differentiation and proliferation of cells by growth factor stimulation, phosphorylation of PLSCR1 by Src-kinase translocated it to the nucleus where it was shown to up regulate IP3R1 gene expression [12]. PLSCR1 is also substrate for other kinases such as IgE receptor tyrosine kinase, c-Abl kinase indicating it functions as a signaling molecule involved in diverse signaling pathways [13]. Interferon has been shown to up regulate hPLSCR1 expression and is considered as an amplifying factor in antiviral responses [14]. hPLSCR1 has also been shown to interact with multiple other proteins which include onzin, ECM1, HCV proteins EI and E2, CD1 and 4 cellular receptors thereby regulating their functions [15], [16], [17], [18].

In variety of cancer cells variable expression of hPLSCR1 was observed notably in myeloid leukemia where its expression was down regulated [19]. In the myeloid leukemic U937 cells inducible expression of PLSCR1 arrested the proliferation of cells at G1 phase implicating the antileukemic role of PLSCR1. Induction of PLSCR1 increased expression of cyclin dependent kinase proteins such as p27 and p21 and down regulation of S-phase proteins such as SKP2. Antiapoptotic proteins such as c-myc and Bcl-2 were also down regulated upon PLSCR1 induction [19]. However despite the progress made in understanding the antileukemic role of hPLSCR1, the exact mechanism by which PLSCR1 down regulation occurs in such cancer cells remains unknown. In this study, we hypothesize that Snail TF might bind to promoter region of hPLSCR1 and regulate its expression based on bioinformatic studies. Our results clearly showed that Snail transcription factor binds to PLSCR1 promoter and down regulates its expression suggesting its possible implications in cancer therapy.

Section snippets

Cell culture

Hek-293, IMR-32 cells were maintained in DMEM and RPMI-160 (Hyclone, Logan, UT, USA) medium supplemented with 10% and 20% FBS respectively and antibiotics at 37 °C and 5% CO₂. O-Nitrophenyl-β-d-galactopyranoside (ONPG), Adenosine triphosphate (ATP) and d-luciferin were obtained from Sigma Aldrich (USA).

Plasmid construction

Snail transcription factor cDNA was directionally cloned between BamH1 and Xho1 sites of pCDNA vector which contains N-terminal FLAG tag. Sequencing was done to verify the construct; expression of

Identification of transcriptional factor binding sites

To identify the cis-acting elements within the hPLSCR1 promoter, preliminary screening was done using ConSite, open source software that predicts the binding sites for known TFs. Fig. 1A shows the putative TFs binding sites within the hPLSCR1 promoter region which include Snail, AML-1 and Sox-5. The identified putative TFs binding sites by ConSite were performed using a cut of score of 100%. The putative binding site for the Snail TF within the promoter was CAGGTG which was similar to the Snail

Discussion

hPLSCR1 a multifunctional protein shows variable expression in a variety of cancers. In ovarian cancer cell line HEY 1B, increased expression of hPLSCR1 was observed to enhance the invasive properties in ovarian cancer cells [21]. It has also been reported that over expression of hPLSCR1 in athymic nude mice was associated with marked suppression of tumor development, increased infiltration of macrophages and also promoted differentiation. In the myeloid leukemia cell line U937, over expression

Conclusions

To conclude we show for the first time transcription regulation of hPLSCR1 by Snail TF and also confirmed the predicted Snail binding site within hPLSCR1 promoter. Interaction of Snail with hPLSCR1 promoter may have profound impact in variety of cellular processes notably as to how cancer cells escape apoptosis.

Acknowledgment

Vincent Gerard Francis acknowledges CSIR (India) for providing fellowship and IIT-Madras for funding.

References (30)

S.K. Sahu et al.
Phospholipid scramblases: an overview
Arch. Biochem. Biophys.
(2007)
Q. Zhou et al.
Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1
Blood
(2002)
M.H. Chen et al.
Phospholipid scramblase 1 contains a nonclassical nuclear localization signal with unique binding site in importin alpha
J. Biol. Chem.
(2005)
Q. Zhou et al.
Phospholipid scramblase 1 binds to the promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene to enhance its expression
J. Biol. Chem.
(2005)
M. Nanjundan et al.
Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor
J. Biol. Chem.
(2003)
J. Sun et al.
c-Abl tyrosine kinase binds and phosphorylates phospholipid scramblase 1
J. Biol. Chem.
(2001)
J. Merregaert et al.
Phospholipid scramblase 1 is secreted by a lipid raft-dependent pathway and interacts with the extracellular matrix protein 1 in the dermal epidermal junction zone of human skin
J. Biol. Chem.
(2010)
Q. Gong et al.
Phospholipid scramblase 1 mediates hepatitis C virus entry into host cells
FEBS Lett.
(2011)
M. Herfs et al.
Regulation of p63 isoforms by snail and slug transcription factors in human squamous cell carcinoma
Am. J. Pathol.
(2010)
R. Kalluri et al.
The basics of epithelial–mesenchymal transition
J. Clin. Invest.
(2009)

E.D. Hay

An overview of epithelio-mesenchymal transformation

Acta Anat. (Basel)

(1995)

D.S. Micalizzi et al.

Epithelial–mesenchymal transition in cancer: parallels between normal development and tumor progression

J. Mammary Gland Biol. Neoplasia

(2010)

D. Medici et al.

Snail and Slug promote epithelial-mesenchymal transition through β-catenin-T-cell factor-4-dependent expression of transforming growth factor- 3

Mol. Biol. Cell

(2008)

M.A. Nieto

The snail superfamily of zinc-finger transcription factors

Nat. Rev. Mol. Cell. Biol.

(2002)

J.G. Stout

Isolation of an erythrocyte membrane protein that mediates Ca²⁺-dependent transbilayer movement of phospholipid

J. Biol. Chem.

(1996)

Cited by (12)

The role of human phospholipid scramblases in apoptosis: An overview
2017, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
Preliminary bioinformatics studies reveal that several putative binding sites for Snail were predicted in the promoter of hPLSCR1 and hPLSCR4. EMSA, ChiP assay and knockdown studies confirmed the transcriptional downregulation of hPLSCR1 and hPLSCR4 by Snail [28,29]. Snail was known to suppress TGF-β induced apoptosis and trigger EMT in hepatocytes.
Human phospholipid scramblases (hPLSCRs) are a family of four homologous single pass transmembrane proteins (hPLSCR1-4) initially identified as the proteins responsible for Ca²⁺ mediated bidirectional phospholipid translocation in plasma membrane. Though in-vitro assays had provided evidence, the role of hPLSCRs in phospholipid translocation is still debated. Recent reports revealed a new class of proteins, TMEM16 and Xkr8 to exhibit scramblase activity challenging the function of hPLSCRs. Apart from phospholipid scrambling, numerous reports have emphasized the multifunctional roles of hPLSCRs in key cellular processes including tumorigenesis, antiviral defense, protein and DNA interactions, transcriptional regulation and apoptosis. In this review, the role of hPLSCRs in mediating cell death through phosphatidylserine exposure, interaction with death receptors, cardiolipin exposure, heavy metal and radiation induced apoptosis and pathological apoptosis followed by their involvement in cancer cells are discussed. This review aims to connect the multifunctional characteristics of hPLSCRs to their decisive involvement in apoptotic pathways.
Snail represses the expression of human phospholipid scramblase 4 gene
2016, Gene
Citation Excerpt :
Till date there are no reports suggesting transcriptional regulation of hPLSCR4, and there are two reports on transcriptional regulation of hPLSCR1. Snail was shown to downregulate the expression of hPLSCR1 in IMR-32cells (Francis et al., 2014) and c-Myc was shown to up regulate the expression of hPLSCR1 in HEK 293 cells (Vinnakota and Gummadi, 2016). Online prediction tools suggested a putative Snail binding site on hPLSCR4 promoter.
Human phospholipid scramblases (hPLSCRs) are a group of transmembrane ATP independent lipid transporters mediating bi-directional transport of phospholipids. There are four homologues hPLSCR1-hPLSCR4 and hPLSCR1 is the extensively studied homologue among them. hPLSCR4 shares 48% homology with hPLSCR1 and mediates scrambling of PLs similar to hPLSCR1 in Ca^2 + dependent manner. Transcriptional regulation helps in better understanding of the function and the expression of a protein. Till date there are no reports suggesting the transcriptional regulation of hPLSCR4. In this study, we identified Snail to be a potent regulator of hPLSCR4. ConSite tool predicted the presence of a putative Snail binding site with a consensus sequence of ^− 1521CAGGTG^− 1516 on hPLSCR4 promoter. Luciferase assays depicted a dose dependent decrease in hPLSCR4 promoter activity with an increase in amount of Snail. Deletion analysis revealed that the region from − 1380 to − 2100 to be the regulatory region of hPLSCR4. Knock down studies further confirmed Snail mediated downregulation of hPLSCR4, as the mRNA and the protein levels of hPLSCR4 considerably increased under knock down conditions. The in vivo interaction of Snail with hPLSCR4 promoter was further confirmed by ChIP assay. This is the first report on the transcriptional regulation of hPLSCR4, where Snail was shown to downregulate the expression of hPLSCR4.
Two c-Myc binding sites are crucial in upregulating the expression of human phospholipid scramblase 1 gene
2016, Biochemical and Biophysical Research Communications
Citation Excerpt :
Preliminary analysis of the hPLSCR1 promoter using ConSite revealed the presence of many putative TF binding sites like Snail, Sox-5, and AML-1 at 100% cutoff. Our earlier report showed Snail mediated downregulation of hPLSCR1 in IMR-32 cells [17]. However, there were no c-Myc binding sites predicted at 100% cutoff.
Human phospholipid scramblase 1 (hPLSCR1) is a type II endofacial membrane protein which mediates bi-directional transport of phospholipids across the plasma membrane. hPLSCR1, a multifunctional protein with variety of roles in apoptosis, tumor progression, cell signaling and anti-viral defense. The expression of such a multifunctional protein should be under tight regulation. Apart from a single report showing snail mediated down regulation of hPLSCR1, the molecular mechanisms regulating the expression of scramblases are not well elucidated. In this study we identified c-Myc as a transcriptional regulator of hPLSCR1. Transcription factor prediction tool ConSite predicted three binding sites for c-Myc. Reporter gene assays and western blot analysis revealed c-Myc mediated up regulation of hPLSCR1 expression. Deletion construct −790 lacking one c-Myc binding site showed a 27% decrease in promoter activity while deletion construct −469 lacking two c-Myc binding sites showed a 62% decrease in promoter activity. Site directed mutagenesis revealed the importance of c-Myc binding sites from −751 to −756 and −548 to −553 on the promoter of hPLSCR1in transcriptionally regulating the expression of hPLSCR1. The results were further confirmed by shRNA mediated knock down of endogenous c-Myc and in vivo interactions by ChIP assay.
Roles and regulation of phospholipid scramblases
2015, FEBS Letters
Citation Excerpt :
Although PKC-δ, JNK, and STAT1 signalling molecules as well as the ISRE element present in the untranslated exon 1 of PLSCR1 contribute to the transcriptional induction of PLSCR1, the complete mechanism of its transcriptional regulation remains to be investigated. Thus far, the only transcriptional regulatory molecule identified to regulate PLSCR1 is Snail [54]. Further investigations are required to identify transcriptional regulators of PLSCR1 and other phospholipid scramblase family members.
Phospholipid scramblase activity is involved in the collapse of phospholipid (PL) asymmetry at the plasma membrane leading to externalization of phosphatidylserine. This activity is crucial for initiation of the blood coagulation cascade and for recognition/elimination of apoptotic cells by macrophages. Efforts to identify gene products associated with this activity led to the characterization of PL scramblase (PLSCR) and XKR family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli. Meanwhile, TMEM16 family members were identified to externalize phosphatidylserine in response to elevated calcium in Scott syndrome platelets, which is critical for activation of the coagulation cascade. Herein, we report their mechanisms of gene regulation, molecular functions independent of their scrambling activity, and their potential roles in pathogenic conditions.
Phospholipid scramblase 1: a protein with multiple functions via multiple molecular interactors
2022, Cell Communication and Signaling
Transcription regulation of human phospholipid scramblase 1 (hPLSCR1) by a Sry related high mobility group transcription factor, S-SOX5
2022, Research Square

View all citing articles on Scopus

View full text

Snail interacts with hPLSCR1 promoter and down regulates its expression in IMR-32

Highlights

Abstract

Introduction

Section snippets

Cell culture

Plasmid construction

Identification of transcriptional factor binding sites

Discussion

Conclusions

Acknowledgment

Arch. Biochem. Biophys.

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Am. J. Pathol.

The basics of epithelial–mesenchymal transition

J. Clin. Invest.

An overview of epithelio-mesenchymal transformation

Acta Anat. (Basel)

Epithelial–mesenchymal transition in cancer: parallels between normal development and tumor progression

J. Mammary Gland Biol. Neoplasia

Snail and Slug promote epithelial-mesenchymal transition through β-catenin-T-cell factor-4-dependent expression of transforming growth factor- 3

Mol. Biol. Cell

The snail superfamily of zinc-finger transcription factors

Nat. Rev. Mol. Cell. Biol.

Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid

J. Biol. Chem.

Isolation of an erythrocyte membrane protein that mediates Ca²⁺-dependent transbilayer movement of phospholipid