extfieldsofstudy: Biology Medicine

text:
Protein signatures of molecular pathways in non-small cell lung carcinoma (NSCLC): comparison of glycoproteomics and global proteomics

Background Non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer deaths in the United States. More than half of NSCLC patients have clinical presentations with locally advanced or metastatic disease at the time of diagnosis. The large-scale genomic analysis of NSCLC has demonstrated that molecular alterations are substantially different between adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). However, a comprehensive analysis of proteins and glycoproteins in different subtypes of NSCLC using advanced proteomic approaches has not yet been conducted. Methods We applied mass spectrometry (MS) technology featuring proteomics and glycoproteomics to analyze six primary lung SqCCs and eleven ADCs, and we compared the expression level of proteins and glycoproteins in tumors using quantitative proteomics. Glycoproteins were analyzed by enrichment using a chemoenzymatic method, solid-phase extraction of glycopeptides, and quantified by iTRAQ-LC–MS/MS. Protein quantitation was further annotated via Ingenuity Pathway Analysis. Results Over 6000 global proteins and 480 glycoproteins were quantitatively identified in both SqCC and ADC. ADC proteins (8337) consisted of enzymes (22.11%), kinases (5.11%), transcription factors (6.85%), transporters (6.79%), and peptidases (3.30%). SqCC proteins (6967) had a very similar distribution. The identified glycoproteins, in order of relative abundance, included membrane (42%) and extracellular matrix (>33%) glycoproteins. Oncogene-coded proteins (82) increased 1.5-fold among 1047 oncogenes identified in ADC, while 124 proteins from SqCC were up-regulated in tumor tissues among a total of 827 proteins. We identified 680 and 563 tumor suppressor genes from ADC and SqCC, respectively. Conclusion Our systematic analysis of proteins and glycoproteins demonstrates changes of protein and glycoprotein relative abundance in SqCC (TP53, U2AF1, and RXR) and in ADC (SMARCA4, NOTCH1, PTEN, and MST1). Among them, eleven glycoproteins were upregulated in both ADC and SqCC. Two glycoproteins (ELANE and IGFBP3) were only increased in SqCC, and six glycoproteins (ACAN, LAMC2, THBS1, LTBP1, PSAP and COL1A2) were increased in ADC. Ingenuity Pathway Analysis (IPA) showed that several crucial pathways were activated in SqCC and ADC tumor tissues. Electronic supplementary material The online version of this article (doi:10.1186/s12014-017-9166-9) contains supplementary material, which is available to authorized users.

Background
Comprehensive genomic profiling of primary non-small cell lung carcinoma (NSCLC) has identified mutations of multiple driver genes, especially oncogenes such as AKT1, ALK, EGFR, ERBB2, KRAS, MET, NRAS, BRAF, PIK3CA, RET, ROS1, and others [1,2]. Based on these findings, several clinical trials have been implemented that target these molecular pathways [3][4][5]. For example, therapeutic treatments targeting tumors with EGFR alterations and ALK gene rearrangements have shown improved outcomes [6][7][8]. However, NSCLC continues to be the leading cause of cancer mortality, accounting for approximately 27% of all cancer deaths in the United States. In 2017 alone, it is estimated that over 222,500 patients will be diagnosed with NSCLC and more than 155,870 patients will die from the disease [9]. Thus, it is critical to understand the role of molecular alterations in NSCLC development, progression, and treatment susceptibility.
NSCLC is the most common morphological type of lung carcinoma (85-90%) and it consists of two major histological subtypes (ADC: 40-50%; SqCC: 25-30%) and several other subtypes (5%) [10]. The development and progression of a NSCLC tumor is a multistep process. NSCLC tumors are characterized by aberrant gene and protein expression, which subsequently leads to phenotypic transformation of cells, initiation and progression of the tumor [6][7][8]11]. The large-scale genomic analysis of NSCLC has demonstrated that molecular alterations are substantially different between ADC and SqCC [12,13]. The alterations of EGFR and rearrangement of ALK in ADC are detected in approximately 25% of tumors; loss-of-function mutations in LKB1/ STK11, NF1, CDKN2A, SMARCA4 and KEAP1 are also identified [12]. In contrast, SqCCs rarely harbor EGFR mutations or AKL rearrangement; instead, SqCCs demonstrate alterations in other genes such as RTKs, DDR2 and FGGRs, and inactivated CDKN2A, PTEN, KEAP1, MLL2, HLA-A, NFE2L2, NOTCH1 and RB1 [13].
The complex alterations of genetic pathways are associated with aberrant cellular protein expression patterns. More than 50% of cellular proteins, including secreted, cell surface and intracellular proteins, are glycosylated. Protein glycosylation is known to play critical roles in the regulation of cell growth, differentiation and migration [14][15][16]. Glycoprotein expression may directly reflect the physiological and/or pathological status of the lung parenchyma. Glycosylation in NSCLCs occurs on diverse molecules and involves a large number of genetic and proteomic alterations; thus a single protein biomarker is unlikely to be representative of all NSCLCs. The profiling of proteins and glycoproteins is particularly important for understanding NSCLC biology and identifying candidate molecular markers.
Over the past decade, efforts have been devoted to the identification of protein biomarker candidates in the various forms of lung cancers. For example, more than six hundred articles have been published for predictive lung cancer biomarkers, while over three hundreds publications are related to prognostic biomarkers [17][18][19][20]. These observations demonstrate the general effort and interest in the discovery of potential protein biomarkers for detecting and monitoring the progression of lung cancer. Most of these studies used non-human tissues or body fluid to study genes or proteins; however, few have been focused on the glycoproteins in human lung tissues using hydrazide chemistry to specifically study protein glycosylation. Additionally, the molecular complexity of NSCLC is still not fully understood.
In this study, we focused on profiling the protein and glycoprotein signatures of primary lung SqCC and ADC using advanced proteomics and MS technology, and we compared glycoproteins and proteins in tumor tissues using Ingenuity Pathway Analysis (IPA) (http:// www.ingenuity.com/products/ipa). The purposes of this study are: (1) to profile proteins and glycoproteins in two NSCLC subtypes; (2) to understand their potential roles in molecular signaling pathways; (3) to correlate signature proteins with cellular biological functions and tumor biological pathways, essentially for the discovery of molecular markers; and (4) to provide information regarding the potential indirect molecular targets in several well-known genetic pathways.

Protein distribution in lung tissue
Comprehensive profiling of proteins was performed on 18 lung tissues from normal, healthy control, ADC and SqCC patients. Over 8000 proteins were quantitatively identified in ADC (Fig. 1a) and 6900 proteins in SqCC (Fig. 1b). Many proteins were substantially increased in ADC and SqCC tumor tissues compared to the normal tissues, as shown in Additional file 2: Table S2, Additional file 3: Table S3, Additional file 4: Table S4, Additional file 5: Table S5. Proteins from ADC or SqCC were identically distributed based on their cellular types, and enzymes, transcription factors, transporters, kinases, peptidases, and phosphatases were dominant. Classification based on cellular location (Fig. 2) indicated that half of the proteins were cytoplasmic (47% in ADC and 49% in SqCC). The majority of glycoproteins were localized to the plasma membrane (42%), extracellular space (~33%), and cytoplasm (~20%) (Fig. 2c, d). Over 1000 enzymes were concurrently identified in the lung tissues: 241 proteins that are encoded by transcriptional factors were identified in both subtypes of NSCLC tissues. In lung tissue, about 5% of the proteins were kinases: 5.11% in ADC and 5.05% in SqCC. Among the 228 identified kinases in SqCC tissues, 33 of them were upregulated (>1.5-fold); only 3 kinases were slightly over-expressed and 4 were down-regulated in ADC out of a total of 248 kinases. Most kinases remained stable between 0.67-fold and 1.5-fold.

Quantitative analysis of glycoproteins
We identified over 480 glycoproteins from lung ADC or SqCC tissues; 443 glycoproteins were present in both subtypes (Fig. 1c, d; Additional file 6: Table S6). Glycoproteins, such as DSC3, DSG3, PLOD2, DSC2, VCAN, PLOD1, DSG2, SLC2A1, TIMP1, and EGFR were increased in SqCC. Conversely, glycoproteins including PLOD2, DSG2, PLOD1, DSC2, and TIMP1 were upregulated in ADC tumor tissues. Other glycoproteins were only found to be upregulated in ADC, including FAP, CALU, POSTN, and CEACAM6. The global results showed that CEACAM and MUC were significantly increased in ADC, suggesting that the upstream regulators of those glycoproteins may be strikingly upregulated different from SqCC. However, it is difficult to draw a conclusion only based on the similarity of a single protein or glycoprotein between ADC and SqCC. Instead, a panel of proteins or glycoproteins may be better than an individual entity to represent the diseases. A systematic analysis of protein activation was inferred through statistical interpretation using IPA [21,22].

Enzyme regulation
Enzymes are known to catalyze thousands of biochemical reactions and they are indispensable in the functions of living organisms. Proteins functioning as enzymes in the lung may regulate their biological functions and activate or inhibit diseases. Among them, the abundance of six proteins were substantially increased in both NSCLC subtypes, including BCAT1, UPP1, CARS2, HAT1, CD38, and PSAT1 (Note: protein name is given in the SI). Upregulated in both ADC and SqCC tissues, BCAT1 has been found to promote cell proliferation through amino acid catabolism in the malignant tumor of the glial tissue [23], whereas UPP1 along with other genes are predominantly expressed in the pancreatic ductal epithelium [24]. HAT1, CARS2, CD38, and PSAT1 are upregulated in SqCC but downregulated in ADC. A critical oncogene with roles in protein acetylation, HAT1 has been linked to different types of cancers. The CD38 protein is a marker of cell activation and it is associated with leukemia, myelomas, and solid tumors [25]. Overexpression of PSTA1 can stimulate cell growth and increase the chemoresistance of colon cancer cells [26]. Differential expression of these proteins may trigger varied phenotypes in NSCLC.
The relative abundance of other proteins, such as enzymes, have been exclusively changed in ADC or SqCC. Approximately 40 enzymes are upregulated in ADC tissues, notably ENPP2, LAMB2, TAB1, ASAH1, LAMP2, GPNMB, HSPG2, CTBS, GLA, and MAN1A1. ENPP2 is responsible for the production of lysophosphatidic acid from lysophosphatidylcholine. It has been identified as a potent tumor mitogen, a cell motility stimulating factor, and it plays a role in cell proliferation [27]. Recent studies indicated that overexpression of the ENPP2 gene increases cell tumorigenesis, invasion, and metastases in breast cancer [28]. Inhibition of ENPP2 can delay breast tumor growth and lung metastasis [29]. TAB1 (TGF-beta activated kinase 1) can mediate diverse intracellular signaling pathways, particularly the promotion of TGF-β mediated nuclear factor-κB (NFκB) activation during cancer progression [30]. Several matrix degrading enzymes (MMPs) were down-regulated in lung ADC tissues, resulting in the overexpression of extracellular matrix glycoproteins. An extracellular matrix glycoprotein, Laminin (LAMB2) contains the major non-collagenous constituent of basement membranes. This glycoprotein is involved in many biological processes including cell adhesion, signaling, differentiation, and metastasis [31]. A glycosylated beta subunit, ASAH1 cleaves a mature enzyme post-translationally, whose expression is correlated with improved prognosis in estrogen receptor-positive breast cancer [32]. LAMP2, GPNMB, and HSPG2 are associated with tumor cell metastasis and tumor growth [33][34][35]. Other genes can regulate protein glycosylation, e.g., degradation of asparagine-linked glycans (CTBS) [36], hydrolysis of the terminal alpha-galactosyl moieties of glycoproteins or glycolipids (GLA) [37], or catalysis of the hydrolysis of the three terminal mannose residues of N-glycans (MAN1A1) [38].

Transcriptional regulators
Some transcriptional regulators were substantially decreased (AGAP3, ACTN1, and TSC22D4), while FOXK1 was increased in ADC. An actin binding protein, ACTN1 cytoskeletal isoform is involved in binding actin to the membrane, and reduction of ACTN1 by siRNA can enhance tumor-free survival [39]. Conversely, other transcriptional regulators were increased in lung SqCC tissues, e.g., BTF3, PYCARD, NFκBIE, EDF1, HMGA1, MAX, MTDH, NMI, and LPXN. A transcription factor and a modulator of apoptosis, BTF3 (basic transcription factor 3) can initiate apoptosis and activate NFκB [40]. BTF3 interacts with CARD domain containing proteins such as PYCARD that mediate the assembly of apoptotic signaling complexes, leading to NFκB activation and increased protein expression [41]. EDF1, endothelial differentiation-related factor-1, is an important gene for tissue angiogenesis and cell proliferation [42]. The overexpression of HMGA1 could associate with the metastatic progression of NSCLC cells. In fact, HMGA1 is a non-histone protein that alters chromatin structure and regulates other transcriptional genes by either enhancing or suppressing transcription factors, exemplifying inhibition of the function of p53 family members in thyroid cancer cells [43,44]. Functioning as an oncogene in many cancers and highly expressed in cancers, MTDH assists in cancer progression and development. It is induced by the c-MYC oncogene and plays roles in the anchorageindependent growth of cancer cells [45]. MAX, on the other hand, can form a dimer with c-MYC to promote cancer cell proliferation and normal cell apoptosis [46].
Several transcriptional proteins were detected only in either SqCC (32) or ADC (53) tissues. However, most proteins had negligible regulation (0.67-1.50-fold change) in either lung subtype, except for JUNB and ANKLE2 proteins. JUNB is uniquely overexpressed in ADC and it is involved in regulating gene activity following the primary factor response. JUNB can promote cell invasion and angiogenesis in cancer cell carcinoma [47]. ANKLE2 was upregulated only in SqCC (1.5-fold), indicating the unique characteristic of this transcriptional protein in SqCC.

Protein kinases
Kinases transfer the phosphate groups from high-energy, phosphate-donating molecules to specific substrates, or vice versa. Protein phosphorylation or de-phosphorylation can greatly affect kinase activity, reactivity, and ability to bind other molecules. Kinases are thus essential in metabolism, cell signaling and other cellular pathways. Many protein kinases play roles in cell metabolism, including NADK, PKM, NAGK, PDK3, and ALDH18A1. Up-regulated in SqCC, AK4 (acetylate kinase-4; 2.98fold) has been identified as a marker of poor clinical outcomes in NSCLC, and it can promote cancer metastasis via downregulation of the transcription factor ATF3 [48]. Other kinases have functions in the ERK, PI3K/Akt and PAK signaling pathways. For example, ZAP70 is a kinase required for association with the Shc adaptor protein and coupling of the activated TCR to the RAS/RAF/ERK signaling pathway [49]. Studies showed that the crosstalk between ERK and PI3K/Akt led to the intervention of cell cycle progression and cell death in carcinoma cells [50]. Overall, those proteins regulate diverse cellular pathways in cancer cell differentiation, transcription, proliferation, and apoptosis, e.g., MAP2K1, PRCKCB, PIK3CG, and TP53RK.

Other highly regulated proteins
Major histocompatibility complex (MHC) proteins were over-expressed in both SqCC (MHC-DRBB1, MHC-B, and MHC-DQB1) and ADC (MHC-A, MHC-DRB1, MHC-C, MHC-DQB1, MHC-DRB3, and MHC-DQA1). Both subtypes of NSCLC have MHC class I and II. Importantly, the antigens of the MHC class I are associated with tumor growth and metastasis [51]. Loss of MHC class II gene and protein expression has been shown to be related to decreased tumor immune-surveillance and poor patient survival [52]. Collagens and extracellular matrix proteins are also highly expressed in SqCC (CTHRC1) and ADC (COL8A1, COL1A1, COL1A2). Over-expressed in ADC tissues only, CEACAM5 and MUC1 have been used as lung ADC markers, indicating that they are specific to this cancer subtype [53].

Oncogenes and tumor suppressor genes
Oncogenes and tumor suppressor genes (TSG) may be associated with the onset and progression of tumors. TSG or oncogene mutations could trigger a loss or reduction in cell functions, resulting in aberrant cell cycle progression. Also discussed previously, we found that many TSG-coded proteins are regulated in lung cancers [54][55][56][57]. Hence, it is reasonable to focus our analysis on those proteins whose genes are oncogenes or TSGs. As shown in Fig. 3A, we identified about 327 oncogenes (5%) and 563 TSGs (8%) in SqCC tissues, and 1047 oncogenes (12%) and 680 TSGs (8%) in ADC tissues (SI). There were 102 upregulated oncogene-coded proteins (>1.5-fold) and 46 downregulated TSG-coded proteins (<0.67-fold); 82 oncogenes were overexpressed and 41 TSGs were down-regulated (Fig. 3A). These genes were further studied by pathway analysis to determine their relationship with transcription regulators and how they correlate to diseases (Fig. 3B, C). Table 1 and Additional file 1: Table S1 list the upstream regulators in lung cancers and their regulated oncogenes/TSGs (IPA analysis). Three observations are evident: (a) activation in SqCC and ADC, (b) inhibition in SqCC and ADC, and (c) activation in only one of the subtypes. For example, ERBB2 is activated in both SqCC (z-score = 1.35) and ADC (z-score = 0.45); TP53 is inhibited in both lung cancer subtypes (−0.70; −2.32); and NFκBIA is inhibited in SqCC (−1.49) while it is activated in ADC (0.45). The upstream regulators (Additional file 1: Table S1) are plotted in Fig. 3Ba which compares major transcriptional regulators in SqCC and ADC. In general, most transcriptional regulators are activated in SqCC but inhibited in ADC. Further, we illustrated the downstream regulation of this transcription factor

Table 1 Comparison of oncogenes and tumor suppressor genes (TSGs) on lung SqCC and ADC tumors
The activation or inhibition is based on quantitative data from proteins and glycoproteins. The analysis was performed using Ingenuity Pathway Analysis (IPA). NC no change, ND not detected, NE no effect, "+" activation or upregulation, "−" inhibition or downregulation, "*" only detected in glycoproteins by SPEG. Gene mutation is compared with studies using mRNA, microRNA, and DNA sequencing on ADC [12] and SqCC [13] Fig. 3Bb-d). TP53 inhibition causes the differential regulation of genes in ADC and SqCC. For example, TP53 inhibition downregulates CAT, CAV1, CDKN1B, CNN1, CTGF, and CXCL12 in both ADC and SqCC (Fig. 3Bb); it increases the expression of TOP2A, COL1A2, and MCM2 in ADC, and it reduces the expression of ZYX, ANXA1, ASS1, CD82, DKK3, FAS, NDRG2, and PTPN11 in SqCC. Protein expression may be related to a variety of diseases and biological functions in cells. Figure 3Ca lists the effect of protein regulation on different diseases and biological functions. The x-axis displays the diseasebiological functions and the z-score is indicated on the y-axis. The diseases or biological functions are activated when Z > 0 and they are inhibited when Z < 0. To illustrate the results, a few examples are used for inversely regulated (Fig. 3Cb) and concurrently increased (Fig. 3Cc) biological functions. SqCC cytoplasm development is inhibited by a set of oncogenes (TNC, TAC-STD2, KSR1, EGFR, CDK5) and TSGs (DMD, CXCL12, CDGF, CDKN1B, CDH13, CAVS, VIL1). ADC cytoplasm development is activated by oncogenes (JUNB, BOP1) and TSGs (ANXA1, CD44, PTPN11, TSC1, ZYX). The proliferation of tumor cells is activated by different sets of oncogenes and TSGs in both SqCC and ADC (Fig. 3Cc). Whether these genes are responsible for the specific diseases or biological functions needs to be further validated.

NSCLC signaling in SqCC and ADC
IPA analysis of proteins and glycoproteins reveals whether a signaling pathway is activated in SqCC and ADC. How the molecular markers are regulated in NSCLC signaling can be evaluated using proteins identified from SqCC and ADC (benign vs. cancer). Figure 4 shows the molecular markers that regulate the biological functions in lung cancers. The proteins are listed by their cellular location such as extracellular matrix, cytoplasm, and nucleus. In benign SqCC (Fig. 4a), EGFR (or HER1) is down-regulated and ERBB2 (or HER2) is upregulated by extracellular matrix proteins. EGFR downregulation leads to the overexpression of GRB2 (inhibition effect) or downregulation of PLCγ1 (activation effect). Together with an activation effect by K-Ras (down-regulated in SqCC normal), c-Raf is down-regulated and eventually inhibits cell proliferation. Although EGFR is upregulated in SqCC cancer, the same signaling and downstream regulation is observed, indicating that regulation of EGFR alone may not affect cell proliferation or apoptosis (Fig. 4a, b).
TSGs (TP53, FHIT) show dramatically different effects on cell growth arrest, genomic instability, and apoptosis  (Fig. 4a, b). In SqCC normal tissue, p53 has no observable effect on cell diseases or functions. However, it is drastically activated in SqCC cancer, leading to the activation of cell apoptosis. Numerous studies have demonstrated that the increased expression of p53 mutation was associated with primary lung cancer [58,59] and SqCC in the head and neck [60]. In contrast to p53, FHIT is overexpressed in normal SqCC, activating alveolar or bronchiolar epithelial cells. The deletion of this gene has been found in primary effusion lymphoma cell lines [61]. Along with K-Ras and PTEN, FHIT is genetically altered in ADC and SqCC tumors [62]. These results may indicate the importance of studying TSGs.
NSCLC signaling pathways in ADC tissues have differentially regulated protein signatures in response to changes of the extracellular matrix proteins. Different from SqCC, EGFR is overexpressed in normal ADC, but it is down-regulated in cancerous ADC (Fig. 5a, b). Most molecular signatures are increased in ADC normal tissues, potentially leading to cell proliferation, whereas the opposite phenomenon occurs in ADC tumor tissues (Fig. 5c, d). The overexpression of K-Ras in normal ADC also indirectly downregulates MST1, resulting in the increased incidence of apoptosis. However, MST1 is upregulated in ADC tumor through the reduced expression of K-Ras. Likewise, the highly increased expression of PDK1 and AKT in non-cancerous patients causes the downregulation of BAD, which further leads to decreased apoptosis. In ADC tumors, both MST1 and BAD are upregulated by their upstream regulators, resulting in increased apoptosis. Particularly, p53 remains stable in ADC; however, FHIT shows inhibition in alveolar/bronchiolar epithelial cells. Cell cycle progression and apoptosis are inhibited in ADC tissues.
RXR, retinoid X receptor, exhibits different effects in SqCC and ADC. RXT expression remains stable in ADC; however, the expression of RXR increases in SqCC. As a tumor suppressor, the increased RXR is expected to reduce tumor growth and progression. This result may suggest that the RXR gene may not be the dominant tumor suppressor gene in lung SqCC. Other tumor suppressors including p53 or FHIT may play dominant roles in lung SqCC.

Regulation of protein glycosylation by oncogenes or TSGs
Mutation of oncogenes or TSGs can regulate protein glycosylation whose aberrant modification could associate with diseases. The oncogenes (Additional file 2: Table  S2, Additional file 3: Table S3) or TSGs (Additional file 4: Table S4, Additional file 5: Table S5) that we identified in the current study might affect the expression of glycoproteins in SqCC or ADC. We studied the effect of several key proteins on glycoprotein expression, including oncogenes (ERBB2, MYC, and EGFR) and tumor suppressors (NFκBIA, STAT3, TP53). Figure 6 shows the regulation of glycoproteins by oncogenes or TSGs in SqCC and ADC. The results from the quantitative analysis of glycoproteins can be found in Additional file 6: Table S6. MYC and ERBB2 are activated in both SqCC and ADC, while TP53 is inhibited. EGFR is only activated in SqCC but it is inhibited in ADC. Similar patterns were observed for STAT3 and NFκBIA which were inhibited in SqCC and activated in ADC.
Glycoproteins in the extracellular matrix space are important for indicating diseases and they are likely secreted molecules in circulating body fluids [63]. These glycoproteins can be regulated by single or multiple transcriptional factors, kinases or enzymes. Despite different subtypes, extracellular glycoproteins such as TIMP1, LCN2, TNC, COL3A1, CPD, FOMOD, POSTN, VCAN, THBS2, LTF, and PLTP are useful for the identification of NSCLC or its specific subtypes. On the other hand, ELANE and IGFBP3 are only overexpressed in SqCC (Fig. 6a), while ACAN, LAMC2, THBS1, LTBP1, PSAP, and COL1A2 are uniquely upregulated in ADC. Other glycoproteins in the plasma membrane or cytoplasm are differentially regulated by transcriptional factors (MYC, STAT3, TP53, and NFκBIA). Interestingly, some glycoproteins in Fig. 6 are also oncogenes or tumor suppressors, such as TNC and COL1A2. TNC (tenascin-C) was overexpressed in NSCLC, leading to downregulation of the functions of infiltrating lymphocytes and implication in tumor progression [64].
Our results indicate that MYC activates TNC, but TP53 inhibits its expression (Fig. 6). In fact, TNC degradation is associated with tumor recurrence in early stage in NSCLC [65]. Collagen (COL1A2), together with other proteins that are overexpressed only in ADC, may be specific to ADC subtypes since it is deactivated in lung SqCC.

Conclusion
Profiling of global proteins and glycoproteins could be an effective method for understanding molecular signaling pathways and the regulation of proteins that are associated with diseases and biological functions. Signature proteins may be useful for predicting disease onset and progression. Based on quantitative analysis of those proteins, we identified the corresponding genes that contain the instructions for producing the proteins. However, studies on genes alone do not necessarily shed light on how their proteins are post-translationally modified. Glycoprotein analysis can investigate beyond genomics and provide information regarding protein expression. Therefore, quantitative analysis of proteins and glycoproteins is essential for discovery of molecular markers.
In this study, the global proteins and glycoproteins from 18 lung tissues were analyzed. Over 6000 global proteins were quantified from both SqCC and ADC, and about 480 glycoproteins were identified from these tissues. Several proteins including enzymes, kinases, or transcription factors were overexpressed in SqCC and ADC, suggesting their common contributions in those subtypes. Many proteins are exclusively regulated in SqCC or ADC: ENPP2, LAMB2, TAB1, GPNMB, and FOXK1 are associated with ADC, while NFκBIE, EDF1, HMGA1, and LPXN are exclusively overexpressed in SqCC. Therefore, it is possible to use these genes to identify upstream regulators for targeted treatment, whereas the overexpressed glycoproteins can be further studied in lung bronchoalveolar lavage (BAL) for discovery of biomarkers for early detection. Further validation of these proteins can be used to identify a molecular panel for lung cancer, thereby increasing the accuracy of early stage detection.
Assuming that the protein level is proportional to gene expression, IPA analysis could determine the upstream regulators and identify the disease-associated proteins in NSCLC [21]. We have used protein data to correlate gene expression to the biological functions and diseases.
Bioinformatics analysis of proteins and glycoproteins explores the essential relationship among proteins and glycoproteins.

Materials and reagents
All chemicals and reagents were purchased from Sigma Aldrich (St Louis, MO) unless specified otherwise. C18 Solid-phase extraction (SPE) cartridges (3 cc Vac Cartridge, 500 mg sorbent) were from Waters Corporation (Milford, MA). Peptide-N-glycosidase F (PNGase F) and denaturing buffer (10×) were from New England Biolabs (Ipswich, MA). Trypsin gold was from Promega (Madison, WI). Quantitative analysis of peptides was performed on a Q-Exactive mass spectrometer (Thermo Scientific, Waltham, MA). Human lung tissues were collected from the Department of Pathology with the approval of the Institutional Review Board of the Johns Hopkins University.

Tissue protein analysis
A total of 18 patient tissues were collected, consisting of one normal tissue from a healthy control (HC), 3 cases of SqCC and tumor-matched benign tissues, and 6 cases of ADC and 5 tumor-matched benign tissues ( Table 2). The collected tissues were dissected using razor blade before being sonicated in an ice-cooled bath for protein  (Fig. 7). Protein concentration was measured by BCA assay (Pierce, Rockford, IL). Four mg proteins from each tissue were added to the final volume of 1 mL (8 M urea in 1 M NH 4 HCO 3 , pH 8.0) [63,66]. Proteins were reduced in 10 mM of tris (2-carboxyethyl) phosphine hydrochloride (TCEP; 20 µL at 0.5 M) at 37°C for 1 h, followed by alkylation using 40 mM of iodoacetamide (IAA) for 1 h at room temperature in the dark. The same amount of TCEP (40 mM) was added to quench excess IAA. Samples were then diluted fivefold (volume) with DI water to contain 1.6 M urea for tryptic digestion (37°C, overnight; trypsin vs. protein = 1:40). The acidified peptides (~0.1% TFA) were cleanup by C18 SPE column (5×, 0.1% TFA) and eluted using 60% ACN (500 µL × 2, 0.1% TFA). Peptide concentration was determined using BCA and peptides were stored at −80 °C prior to use. One mg peptides from each sample were labeled with 4-plex iTRAQ (AB SCIEX, Framingham, MA). The iTRAQ labeled peptides were pooled for C18 cleanup and 10% (~200 µg) of the pooled peptides was chromatographically separated to 24 fractions by basic reverse phase liquid Fig. 7 Schematic diagram of lung tissue specimens using for proteomic analysis. The dissected tissues in lysis buffer were sonicated to extract proteins. After 4-plex iTRAQ labeling, 10% of the peptides were analyzed for global proteins and 90% of the peptides were used for enrichment of glycosite-containing peptides by Solid-phase extraction of N-linked glycopeptides (SPEG) chromatography (bRPLC) on an 1220 Infinity LC system with a Zorbax Extend-C18 analytical column (1.8 µm particles, 4.6 × 100 mm; Agilent Technologies, Inc., CA) [66]. The remaining iTRAQ-peptides (~1.8 mg) were enriched for glycosite-containing peptides using hydrazide chemistry [67]. One µg of the enriched formerly glycosite-containing peptides were analyzed by LC-MS (Additional file 7, Additional file 8, Additional file 9, Additional file 10).

MS data analysis
The MS/MS spectra were directly searched using the SEQUEST search engine [Thermo Proteome Discoverer 1.4.0.288 (PD)] against the NCBI Homo Sapiens database (Download, August 2014). Carbamidomethylation of cysteine residues was set as a fixed modification; oxidation of methionine and deamidation (only for SPEG) of asparagine were set as variable modifications; N-termini and lysines were set as iTRAQ 4-plex fixed modifications. Maximum missed cleavages using trypsin were set to 2 and the minimum peptide length was 7. The search filter was set as follows: at least 1 peptide per protein and 1% FDR for PSM cutoff. The precursor mass tolerance was 10 ppm, while the mass tolerance of fragment ions was 20 ppm. Quantitation was performed using reporter ion intensity of peptides in PD. The filter for PD was set as at least one peptide per protein and high confidence. The quantification was performed using unique peptides; all unique peptides for the same proteins were taken into account for the comparison of protein abundance. Normalization was conducted using the median intensity of all reporter ions. The ratio (iTRAQ) was determined by comparing with the control (114). We used 1.5-fold change (upregulation) or 0.67-fold change (downregulation) as a cut off for biological significance based on the standard deviation and the normalized peptide ratios.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
An environmental pollutant, 9,10‐phenanthrenequinone, activates human TRPA1 via critical cysteines 621 and 665

Abstract Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold, mechanical stimulation, and irritant chemicals. In our recent study, 9, 10‐phenanthrenequinone (9,10‐PQ) is the most potent irritant for activation of NRF2 among 1395 cigarette smoke components and it may be, therefore, important to find its additional targets. Here, we show that 9,10‐PQ functions as an activator of TRPA1 in human embryonic kidney (HEK) cells expressing human wild‐type TRPA1 (HEK‐wTRPA1) and human alveolar A549 (A549) cells. Application of 9,10‐PQ at 0.1–10 μmol/L induced a concentration‐dependent Ca2+ response as well as inward currents at −50 mV in HEK‐wTRPA1 cells. The current response was blocked by TRPA1 antagonists, HC‐030031 (HC) and A‐967079. To test whether 9,10‐PQ affects the cysteine residues of TRPA1, we expressed mutant TRPA1 channels in HEK cells (HEK‐muTRPA1) in which six different cysteine residues were replaced with serine. Among them, a mutation of cysteine 621 (C621S) abolished the 9,10‐PQ‐induced Ca2+ and current responses. The channel activity induced by 9,10‐PQ was also abolished in excised inside‐out patches isolated from HEK‐muTRPA1 cells with the C621S substitution. Although a mutation of cysteine 665 (C665S) reduced the 9,10‐PQ‐induced response, channel sensitization by pretreatment with Cu2+ plus 1,10‐phenanthroline and by internal dialysis of 3 μmol/L Ca2+ restored the response. However, a double mutant with C621S and C665S substitutions had little response to 9,10‐PQ, even when sensitized by Ca2+ dialysis. In A549 cells, 9,10‐PQ induced an HC‐sensitive Ca2+ response. Our findings demonstrate that 9,10‐PQ activation of human TRA1 is dependent on cysteine residues 621 and 665.

Introduction
A chemical compound, 9,10-PQ, which is one of the active components in cigarette smoke and other environmental pollutants, affects a number of mammalian physiological functions via activation of a transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2), production of hydrogen peroxide, and/or the covalent modification of cysteine residues of proteins (Kishikawa et al. 2010;Asahi et al. 2014;Sekine et al. 2016). Treatment of cells with 9,10-PQ, therefore, causes nonacute biological responses such as antiproliferation, inflammation, and apoptosis (Matsunaga et al. 2010(Matsunaga et al. , 2012Hatae et al. 2013). Our recent study has revealed that 9,10-PQ is the most potent chemical to activate NRF2 among 1395 mainstream cigarette smoke components (Sekine et al. 2016), suggesting it can be an exogenous bioactive compound that, when inhaled, has multiple potential targets, particularly in the acute phase of the exposure. In particular, 9,10-PQ is 12.6 times more potent than acrolein, which also effectively activates a cationic channel protein, TRPA1 (Andre et al. 2008).
TRPA1, which is activated by noxious cold, mechanical stress, irritant chemicals, and/or some clinical drugs, is widely expressed in neuronal and non-neuronal organs (Story et al. 2003;Jordt et al. 2004;Macpherson et al. 2007;Fajardo et al. 2008;Matta et al. 2008;Nassini et al. 2010;Andersson et al. 2011;Hatano et al. 2013). Transgenic mice lacking TRPA1 are less sensitive to mechanical stimulation, cold stimuli, and tumor necrosis factor ainduced mechanical hyperalgesia, which suggests that TRPA1 is a nociceptor that mediates acute and inflammatory pain (Obata et al. 2005;Bautista et al. 2006;Kwan et al. 2006;Karashima et al. 2009;del Camino et al. 2010;Fernandes et al. 2011). On the other hand, several cysteine residues of TRPA1 are targeted by physiological and nonphysiological electrophilic compounds that activate the channel (Hinman et al. 2006;Macpherson et al. 2007). In particular, a recent study revealed that a cysteine residue at 621 (C621) is critical for channel activation by the electrophilic compounds, and that cysteine 665 (C665) is not only supportive but also important for its activation (Bahia et al. 2016). In contrast, nonelectrophilic compounds such as D9-tetrahydrocannabinol, nicotine, and menthol (Men) activate TRPA1 via different mechanisms (Jordt et al. 2004;Karashima et al. 2007;Talavera et al. 2009). Since numerous structurally unrelated compounds stimulate TRPA1, research should focus on whether the channel is activated by clinical drugs and environmental chemicals.
In the current study, we show that a chemical pollutant, 9,10-PQ, functions as an activator of human TRPA1. A comparison of the 9,10-PQ-induced responses of wildtype and mutant TRPA1 with responses to allyl isothiocyanate (AITC) identified that both C621 and C665 are important for the 9,10-PQ-induced response, but that C621 is most critical for activation. Treatment with 9,10-PQ also effectively activated endogenous TRPA1 in human alveolar A549 cells. Our findings indicate that 9,10-PQ directly activates human TRPA1 via interaction with C621 and C665.

Recombinant expression of TRP channels in HEK cells
Partially confluent HEK cells (40-60% confluency) were transfected with the pcDNA3.1 and pcAc-GFP plasmids containing human TRPA1 and TRPV1 cloned by ourselves, respectively, using lipofectamine 3000 (Thermo Fisher Scientific, Yokohama, Japan). The TRPA1 mutations were constructed by PCR using mutant oligonucleotide primers in which the cysteine residues at amino acid positions 414, 421, 621, 633, 641, or 665 were replaced with serine (Agilent Technologies, Santa Clara, CA), and all constructs were sequenced. All experiments were performed within 48 h of transfection.

Western blotting
HEK cells were lysed in 50 lL lysis buffer ([in mmol/L] Tris-HCl 50 [pH 8.0], NaCl 150, EDTA 5) including 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors (Suzuki et al. 2014). The cell lysates were incubated on ice for 30 min, vortexed every 5 min, then centrifuged at 20,000g for 30 min at 4°C, and each lysate (10 lg protein) was separated on an 8% polyacrylamide gel. Proteins were then transferred to a PVDF membrane. After blocking nonspecific antibodies for 2 h in Tris-buffered saline (TBS) containing 5% skim milk and 0.1% Tween-20, the PVDF membrane was incubated with the primary antibody (goat anti-human TRPA1; Santa Cruz Biotechnology Inc., Dallas, TX; 1:1000 dilution) overnight at 4°C. The membrane was then washed three times with washing buffer (TBS containing 0.1% Tween-20) and exposed to the secondary antibody (IgG-HRP; 1:10,000 dilution). The membrane was washed again, and detection reagents (Merck Millipore, Darmstadt, Germany) were added to generate a chemiluminescence signal. To determine the relative quantity of TRPA1 to b-actin in each sample the membrane was also exposed to a mouse anti-b-actin monoclonal antibody (1:2000 dilution). Finally, detection was carried out using an LAS-3000 mini apparatus (Fujifilm, Tokyo, Japan).

Patch clamp experiments
Whole-cell and single-channel recording experiments were performed as described previously (Suzuki et al. 2014). The resistance of electrodes was 3-5 MΩ when filled with pipette solution. A Cs + -rich pipette solution for wholecell recordings contained (in mM) Cs-aspartate 110, CsCl 30, MgCl 2 1, HEPES 10, and Na 2 ATP 2 (adjusted to pH 7.2 with CsOH). The free Ca 2+ concentration in the pipette solution was adjusted to 0 (1 mmol/L ethylene glycol tetraacetic acid; EGTA), 0.3 (10 mmol/L EGTA and 6.25 mmol/L CaCl 2 ), and 3 lmol/L (10 mmol/L EGTA and 9.51 mmol/L CaCl 2 ) using Ca 2+ -EGTA buffer. When tripolyphosphate (TPP) was used to maintain TRPA1 channel activity, a bathing or a pipette solution contained (in mmol/L) CsCl 140, MgCl 2 1, EGTA 1, HEPES 10, and TPP 2.89 (adjusted to pH 7.2 with CsOH). Membrane currents and voltage signals were digitized using an analogue-to-digital converter (PCI6229, National Instruments Japan, Tokyo, Japan) driven by WinEDRV3.38 for data acquisition and analysis of whole-cell currents and excised outside-out and inside-out single-channel currents (developed by Dr. John Dempster, University of Strathclyde, UK). The liquid junction potential between the pipette and bath solutions (À10 mV) was corrected when aspartate À -rich pipette solution was used. A ramp voltage protocol from À150 mV to +50 mV for 100 ms was applied every 5 sec from a holding potential of À50 mV. A leak current component was not subtracted from the recorded currents. The outside-out and inside-out patch channel activity was estimated to calculate NPOs, where N is the number of channels in the patch and PO is the open probability. A standard HEPES-buffered bathing solution (2.2 Ca SBS [in mmol/L]: NaCl 137, KCl 5.9, CaCl 2 2.2, MgCl 2 1.2, glucose 14, HEPES 10 [adjusted to pH 7.4 with NaOH]) was used. When CaCl 2 was omitted from the 2.2 Ca SBS (0 Ca SBS), 10 mmol/L CsCl was added. All experiments were performed at 25 AE 1°C.

Measurement of Ca 2+ fluorescence ratio
HEK and A549 cells, which were loaded with 10 lmol/L Fura2-AM (Dojindo, Kumamoto, Japan) in the 2.2 Ca SBS for 30 min at room temperature, were superfused with 2.2 Ca SBS for 10 min and then Fura-2 fluorescence signals were measured at 0.1 Hz using the Argus/HisCa imaging system (Hamamatsu Photonics, Hamamatsu, Japan) driven by Imagework Bench 6.0 (INDEC Medical Systems, Santa Clara, CA). Since the efficacy of gene transfection in HEK cells and the TRPA1 expression level in A549 cells was similar but not identical from cell to cell, we collected 50 and 12-38 single cells on one coverslip for analysis in HEK and A549 cells, respectively, and repeated the same experiment with the other coverslips to reduce variation. In each analysis, a whole-cell area was chosen as a region of interest to average the fluorescence ratio.

Data analysis
Data are expressed as the mean AE SEM. Statistical significance between two groups and among multiple groups was examined using paired or unpaired Student's t-tests, and ANOVA or Tukey's test, respectively.

Results
Expression of wild-type and mutant TRPA1 in HEK cells was confirmed using the following three assays as described previously (Suzuki et al. 2014): TRPA1 mRNA transcript expression (not shown), TRPA1 protein expression (Fig. 1A), and TRPA1 channel function . Using HEK-wTRPA1 cells, we examined the effects of 9,10-PQ on TRPA1. As shown in Figure 1B and C, treatment of HEK-wTRPA1 cells with 9,10-PQ at concentrations between 0.003 and 1 lmol/L effectively induced Ca 2+ responses, whereas failed to evoke any Ca 2+ response in control HEK cells (cont, Fig. 1C). To confirm that 9,10-PQ activates TRPA1 channel currents, we applied 0.1-10 lmol/L 9,10-PQ to HEK-wTRPA1 cells superfused with 2.2 Ca SBS in the whole-cell recording mode. As shown in the upper panel of Figure 1D and also in Figure 1E, the exposure to 9,10-PQ induced inward currents at À50 mV, which had a Figure 1. Activation of TRPA1 by 9,10-PQ in HEK-wTRPA1 cells. (A) Wild-type (WT) and mutant TRPA1 proteins (C414S, C421S, C621S, C633S, and C641S) were assayed by western blotting to confirm the protein expression in HEK cells. b-actin levels were assayed as controls. 9,10-PQ induced Ca 2+ responses in a representative HEK-wTRPA1 cell (B) and the peak 9,10-PQ-induced Ca 2+ response (DCa 2+ i [F 340 /F 380 ]) in HEK-wTRPA1 cells (WT, seven independent experiments) and control HEK cells (cont, three independent experiments) was summarized as a concentrationresponse relationship (C). *P < 0.05 by ANOVA. 9,10-PQ-induced membrane currents at À50 mV in a representative HEK-wTRPA1 cell which was superfused with 1 mmol/L EGTA (the upper trace) and 1 mmol/L EGTA plus 2.89 mmol/L TPP (the lower trace) (D) and the peak 9,10-PQ-induced inward currents at À50 mV was summarized in HEK-wTRPA1 cells as a concentration-response relationship (4-9 and 3-9 independent experiments, respectively, in (E) and (F)). Cells were superfused with 2.2 Ca SBS as a bathing solution. Ramp waveform voltage pulses from À150 to +50 mV for 100 msec, shown by vertical dotted lines, were applied every 5 sec. (G-I) The experimental conditions were identical to those demonstrated in (D-F), except superfusion contained 0 Ca SBS and addition of 30 lmol/L HC after cumulative application of 9,10-PQ. At the end of each experiment, 3 lmol/L 9,10-PQ was applied again in 2.2 Ca SBS (4 and 4-6 independent experiments, respectively, in (H) and (I)). dose-independent variable size and were transient even in the presence of 9,10-PQ. When 2.89 mmol/L TPP, a stabilizer of TRPA1 (Kim and Cavanaugh 2007;Suzuki et al. 2014), was included in the pipette solution, application of 9,10-PQ significantly induced inward currents at À50 mV, which were still variable and sometimes transient (lower panel of Fig. 1D and F). In contrast, the 9,10-PQ-induced currents became sustainable upon the removal of extracellular Ca 2+ (0 SBS), although the current size was smaller ( Fig. 1G and H). In the presence of TPP in the pipette solution, the 9,10-PQ-induced inward currents were larger and dose dependent (Fig. 1I). Moreover, these 9,10-PQ-induced currents were abolished with the addition of 30 lmol/L HC, a TRPA1 antagonist, suggesting that 9,10-PQ can activate human TRPA1.
In intact cells, ion channels might be influenced by biologically active factors, which are mainly produced intracellularly. In addition, 9,10-PQ can cause oxidative stress that changes cellular signaling pathways. To reduce the possibility that such factors might be involved in 9,10-PQ-induced channel activation of TRPA1, we next examined whether 9,10-PQ directly activates TRPA1 channels in outside-out patches excised from HEK-wTRPA1 cells. As shown in Figure 2A, application of 0.3-3 lmol/L 9,10-PQ also induced channel activity in the excised patches. The channel conductance at negative potentials was 50.1 AE 4.4 pS (n = 5) and 49.6 AE 3.2 pS (n = 4) in the presence of 1 and 3 lmol/L 9,10-PQ, respectively ( Fig. 2B), and the unit current amplitude at À50 mV (À2.64 AE 0.19 pA and À2.47 AE 0.15 pA by 1 and 3 lmol/L 9,10-PQ, see also Fig. 2B) was similar to that evoked by 30 lmol/L AITC (À2.64 AE 0.28 pA, n = 5) under experimental conditions that were similar to our previous study (Suzuki et al. 2014). Moreover, the channel activity was reversibly inhibited by the addition of HC (Fig. 2C), suggesting that TRPA1 is activated by 9,10-PQ in a membrane-delimited manner. For comparison, we also examined the effects of 9,10-PQ on channel activity in excised outside-out patches where 0.3 lmol/L Ca 2+ was included in the pipette solution without TPP in 0 Ca SBS (Fig. 2D); application of 9,10-PQ at 1 and 3 lmol/L effectively induced channel activity.
The conservative cysteine-to-serine mutations in the predicted N-terminal ankyrin repeats of human TRPA1 (e.g., C414S, C421S, C621S, C633S, and C641S) have revealed that these cysteine residues play a critical role in the activation of the channel by electrophilic agonists, which are able to interact with the sulfide moiety of these residues. We, therefore, examined the interaction of 9,10-PQ with the cysteine residues in TRPA1 using HEK cells transfected with mutant TRPA1 (HEK-muTRPA1) containing one of the following substitutions: C414S, C421S, C621S, C633S, or C641S. Administration of 9,10-PQ at a concentration between 0.3 and 10 lmol/L to HEK-wTRPA1 ( Fig. 4A and F) and HEK-muTRPA1 cells ( Fig. 4B-F) induced substantial inward currents at À50 mV, except HEK-muTRPA1 cells with C414S and C621S substitutions. Even in the presence of higher intracellular Ca 2+ (3 lmol/L), HEK-muTRPA1 cells with the C414S substitution were completely insensitive to 9,10-PQ (Fig. 4F) as well as AITC and Zn 2+ (Hatano et al. 2013), thus, we excluded this mutant from further analyses. In addition, 9,10-PQ had little effect on HEK-muTRPA1 cells with the C621S substitution in Ca 2+ measuring experiments ( Fig. 4G and H). To further confirm the importance of C621 for the 9,10-PQ-induced response, we systematically compared the response of wild-type and mutant TRPA1 with a C621S substitution to 9,10-PQ and AITC in whole-cell ( Fig. 5A-C) and excised inside-out patch (Fig. 5D-F)  C621S substitution responded only to AITC (Fig. 5B, C, E, and F), suggesting that C621 is more important for the response to 9,10-PQ than AITC. On the other hand, although a quinone-related compound, 1,2-naphthoquinone (1,2-NQ), can activate TRPV1 (Kikuno et al. 2006), 10 lmol/L 9,10-PQ had no effect on human TRPV1 ( Fig. 5G and H).
A recent observation demonstrates that C621 is necessary for human TRPA1 reactivity to electrophilic compounds, and that C665 is secondary (Bahia et al. 2016). Therefore, we next examined the effects of 9,10-PQ on HEK-muTRPA1 cells with a C665S substitution. As shown in Figure 6A, application of 0.3-10 lmol/L 9,10-PQ to this mutant induced substantial inward currents at À50 mV. However, the current responses were clearly smaller (Fig. 6B), suggesting that C665 affects the 9,10-PQ-induced response of TRPA1 even though the critical C621 is conserved. To sensitize this mutant channel, we pretreated the C665S HEK-muTRPA1 cells with 5 lmol/L Cu 2+ plus 15 lmol/L 1,10-Pher and then added 3 lmol/L 9,10-PQ. As a control, we first exposed the mutant to 9,10-PQ only in the presence of 1,10-Pher. This Cu 2+ plus 1,10-Pher-induced sensitization recovered the response to 9,10-PQ in C665S HEK-muTRPA1 cells (Fig. 6C and D), while not in the C621S HEK-muTRPA1 cells (Fig. 6E and F), suggesting that C621 is critical and that C665 is supportive for the 9,10-PQ-induced response.
It is well known that intracellular dialysis of high Ca 2+ sensitizes TRPA1 channel activity (Wang et al. 2008). Therefore, we internally applied 3 lmol/L Ca 2+ to both C621S and C665S HEK-muTRPA1 cells and examined the effects of 9,10-PQ on the membrane currents of these cells at À50 mV. Similar to wild-type TRPA1 (Fig. 7A), the mutant TRPA1 with the C665S substitution had a substantial response to 9,10-PQ ( Fig. 7B and D). Although the mutant TRPA1 with the C621S substitution also responded to 9,10-PQ, the response was relatively small ( Fig. 7C and D); hence, we next examined the effects of 9,10-PQ on a double mutant TRPA1 where both C621 and C665 were replaced with serine. When this mutant TRPA1 was sensitized by 3 lmol/L Ca 2+ , 0.3-10 lmol/L 9,10-PQ induced A96-sensitive small inward currents less than À70 pA at À50 mV in five of seven cells (29.7 AE 14.1 pA, upper trace in Fig. 8A). In two cells, however, 10 lmol/L 9,10-PQ induced A96-sensitive inward currents larger than À900 pA (1422 AE 484 pA, lower trace in Fig. 8A). In contrast, the vehicle-induced inward currents at À50 mV were negligible under the same experimental conditions (Fig. 8C and D). Taken together, at relatively low concentrations 9,10-PQ mainly targets TRPA1 C621 and at higher concentrations 9,10-PQ may have additional effects on sensitizing TRPA1 even without C621. On the other hand, although C665 plays an important role in 9,10-PQ induced activation of TRPA1, it is unlikely that C665 is a target for 9,10-PQ.
Since 9,10-PQ is commonly inhaled in the air, airway organs are potential primary targets of 9,10-PQ. Therefore, we used human alveolar A549 cells to examine whether 9,10-PQ can activate endogenous TRPA1 expressed in human cells. As shown in Figure 9A, application of 10 lmol/L 9,10-PQ to A549 cells evoked a Ca 2+ response in the absence of HC. Since the response was effectively inhibited by preadministration of 30 lmol/L HC (Fig. 9B), A549 cells express TRPA1 which can be a potential target of 9,10-PQ acutely administrated.

Discussion
In the present study, we show that the environmental pollutant 9,10-PQ potently activates human TRPA1 and might function as an irritant when inhaled. In addition, the activation of TRPA1 by 9,10-PQ is dependent on the cysteine residues at 621 and 665 in the N-terminus of the channel. Application of 9,10-PQ also induced activation Figure 2. Activation of TRPA1 channels by 9,10-PQ in excised outside-out patches. (A) A representative 9,10-PQ-evoked single-channel current trace at À50 mV in a patch excised from a HEK-wTRPA1 cell (left panel) and the summary of channel activity of NPO (0.3-3 lmol/L 9,10-PQ, 3-7 independent experiments) (right panel). Each patch was superfused with 2.2 Ca SBS and dialyzed with CsCl-rich pipette solution including 2.89 mmol/L TPP. Vertical lines indicate application of ramp pulses. *P < 0.05 by paired Student's t-test. (B) A representative 9,10-PQ-evoked single-channel current trace by a stepwise change of voltage from À50 mV to À20 mV in a patch excised from a HEK-wTRPA1 cell (left panel) and a summary of the relationship between the current and voltage of 1 and 3 lmol/L 9,10-PQ-evoked single-channel currents (3-5 independent experiments) (right panel). In the lower panel of the trace (B), time and current scales were expanded. Each patch was superfused with 2.2 Ca SBS and dialyzed with CsCl-rich pipette solution including 2.89 mmol/L TPP. The "c" indicates the channel closing level, and "O1" and "O2" represent the first and second channel opening levels, respectively. (C) Inhibitory effects of HC on 9,10-PQ-evoked TRPA1 channel activity in excised outside-out patches. An original current trace (lower panel) and averaged current amplitude for every 3 sec (upper panel) are shown at the left. While 1 lmol/L 9,10-PQ was present in 2.2 Ca SBS, 30 lmol/L HC was added. The relative current amplitude before and during HC application, and after washout, is summarized in the right panel (four independent experiments). Each patch was superfused with 2.2 Ca SBS and dialyzed with CsCl-rich pipette solution including 2.89 mmol/L TPP. *P < 0.05 by Tukey's test. (D) A representative 9,10-PQ-evoked single-channel current trace at À50 mV in a patch excised from a HEK-wTRPA1 cell and the summary of the channel activity of NPO (four independent experiments). Each patch was superfused with 0 Ca SBS and dialyzed with Cs-aspartate-rich pipette solution including 0.3 lmol/L Ca 2+ . Vertical lines indicate application of ramp pulses. *P < 0.05 by paired Student's t-test. Bars = SEM. of endogenous TRPA1 in A549 cells derived from human alveoli, suggesting that environmental 9,10-PQ may cause acute biological actions via activation of TRPA1.
In this study, we clearly showed that 9,10-PQ, an environmental pollutant included in cigarette smoke extract (CSE) and diesel exhaust particles (DEP), is an effective activator of human TRPA1. Under all experimental conditions, 1 lmol/L 9,10-PQ sufficiently activated wild-type human TRPA1. Indeed, CSE, DEP, wood smoke particulate material (WSPM), and insoluble coal fly ash particles can activate TRPA1 (Andre et al. 2008;Deering-Rice et al. 2011Shapiro et al. 2013), and some putative active chemicals composed of these components were identified: crotonaldehyde and acrolein in CSE; ditert-butylphenols in DEP; and agathic acid, coniferaldehyde, and perinaphthenone in WSPM. Moreover, additional TRPA1 agonistic compounds might be generated in the lung from polycyclic aromatic hydrocarbons inhaled via cytochrome P450 (P450) (Lanosa et al. 2010). Among these chemicals, acrolein was relatively more potent and its EC 50 for the activation of rat and human TRPA1 expressed in HEK cells was 0.8-5 lmol/L Andre et al. 2008). In our recent study, 9,10-PQ was the most potent activator among 1395 CSE components using an Figure 3. Comparison of TRPA1 channel activation by 9,10-PQ with conventional TRPA1 agonists and structurally related compounds. Cells were superfused with 0 Ca SBS and dialyzed with Cs-aspartate-rich pipette solution containing 0.3 lmol/L Ca 2+ . Ramp waveform voltage pulses from À150 to +50 mV for 100 msec were applied every 5 sec. Effects of 9,10-PQ (A, 1-10 lmol/L), AITC (B, 3-30 lmol/L), Men (C, 10-300 lmol/L), and Zn 2+ (D, 1-10 lmol/L) on membrane currents at À50 mV in each HEK-wTRPA1 cell. After the final concentration of each agonist was applied, 30 lmol/L HC (black rectangles) was added to block TRPA1 channel current components. As a comparison, 10 lmol/L 9,10-PQ was applied in (C) and (D). The chemical structure of 9,10-PQ is illustrated in the inset of (A). (E) The peak agonist-induced inward currents at À50 mV were pooled and summarized as a concentration-response relationship (3-7 independent experiments). The x-axis indicates concentration of each compound. *P < 0.05 by ANOVA. (F-H) The experimental conditions were identical to those demonstrated in (A-E) except for the application of 1-10 lmol/L 1,10-P-5,6-D (five independent experiments) (F) and 1-10 lmol/L Phenan (five independent experiments) (G). Chemical structures of 1,10-P-5,6-D and Phenan are illustrated in the insets of (F) and (G), respectively. (H) The peak compound-induced inward currents at À50 mV were pooled and summarized as a concentration-response relationship. The x-axis indicates concentration of each compound. *P < 0.05 by ANOVA. N.S. shows no significance. Bars = SEM. 2017 | Vol. 5 | Iss. 4 | e00342 Page 8 antioxidant response element assay in human bronchial epithelial BEAS-2B cells and, furthermore, 9,10-PQ was 12.6 times more effective than acrolein in this assay (Sekine et al. 2016). Although it is difficult to measure the exact amounts of these chemicals in CSE and DEP, 9,10-PQ can be one of the most important chemicals in pollutants as an irritant that affects human health.
Among the TRPA1 agonists employed in this study, 9,10-PQ was the most potent, although accurate comparison of agonistic activity among chemicals is difficult. As shown in Figure 1, inclusion of Ca 2+ in the bathing solution caused potentiation and desensitization of 9,10-PQ-induced TRPA1 channel currents, as previously reported (Doerner et al. 2007;Zurborg et al. Figure 4. Comparison of 9,10-PQ-induced TRPA1 channel activation among mutants in TRPA1 N-terminal cysteine residues. Cells in which a cysteine residue of wild TRPA1 (WT, A) was mutated with serine at 421 (C421S, B), 621 (C621S, C), 633 (C633S, D), and 641 (C641S, E) were superfused with 0 Ca SBS and dialyzed with Cs-aspartate-rich pipette solution including 0.3 lmol/L Ca 2+ . Ramp waveform voltage pulses from À150 to +50 mV for 100 msec were applied every 5 sec. 9,10-PQ was commutatively applied to each mutant to confirm effects on membrane currents at À50 mV. After application of the final concentration of 9,10-PQ, 30 lmol/L HC (black rectangles) was added to block TRPA1 channel current components. (F) The peak 9,10-PQ-induced inward currents at À50 mV were pooled and summarized as a concentration-response relationship (3-7 independent experiments). In HEK-muTRPA1 cells with a C414S substitution, 3 lmol/L Ca 2+ was intracellularly dialyzed to potentiate TRPA1 channel currents. *P < 0.05 by Tukey's test in the presence of 10 lmol/L 9,10-PQ (C621S vs. WT, C421S, C633S, and C641S). (G) and (H) Ca 2+ responses of mutant TRPA1 with C621S to 9,10-PQ. A representative 9,10-PQ-induced Ca 2+ response was shown in a HEK-wTRPA1 (G, upper panel) and a HEK-muTRPA1 with C621S cell (G, lower panel). The peak 9,10-PQ-induced Ca 2+ response (DCa 2+ i [F 340 /F 380 ]) was summarized as a concentration-response relationship for WT TRPA1 (3 independent experiments) and mutant TRPA1 with C621S (3 independent experiments). To confirm the channel expression, 30 lmol/L AITC was applied at the end of experiment. *P < 0.05 by ANOVA. Bars = SEM. 2007; Wang et al. 2008). It is, therefore, notable that using pharmacological assays to measure TRPA1 agonist-induced Ca 2+ may have limitations. On the other hand, TRPA1 whole-cell currents were sustainably and cumulatively activated by application of 9,10-PQ and AITC in the absence of extracellular Ca 2+ , suggesting that these are direct activators of human TRPA1 with little dependency on Ca 2+ , and 9,10-PQ is more potent than AITC. In contrast, under the same experimental conditions, the agonistic activities of Zn 2+ and Men were weak and negligible, respectively. Since inclusion of 3 lmol/L Ca 2+ in the pipette solution increased the response to 10 lmol/L Zn 2+ (52.0 AE 5.2 pA, n = 3 vs. 339.7 AE 24.9 pA, n = 4 in the presence of 0.3 and 3 lmol/L Ca 2+ , respectively, at À50 mV) and 300 lmol/L Men (6.25 AE 6.25 pA, n = 4 vs. 339.7 AE 24.9 pA, n = 4 in the presence of 0.3 and 3 lmol/L Ca 2+ , respectively, at À50 mV), Zn 2+ and Men could be TRPA1 agonists in a Ca 2+ -dependent manner. In fact, 1 lmol/L Zn 2+ and 250 lmol/L Men are substantially effective in the elevation of intracellular Ca 2+ in HEK-wTRPA1 cells (Xiao et al. 2008;Hu et al. 2009).
Although superoxide and hydroxyl radicals are generated by enzymatic one-electron reduction of 9,10-PQ by P450 reductase within 30 sec of treatment (Sugimoto et al. 2005), it is unlikely that intracellular factors are involved in 9,10-PQ-induced activation of TRPA1. Based on the evidence that application of 9,10-PQ to outside-out (Fig. 2) and inside-out patches (Fig. 5D) excised from HEK-wTRPA1 cells effectively induced HC-sensitive channel activity, we concluded that a membrane-delimited pathway must be involved in the activation. On the other hand, it has been shown that polyphosphates are required for electrophilic channel activity, which stabilizes the coiled-coil domains in the intracellular C-terminus (Kim and Cavanaugh 2007;Paulsen et al. 2015). Since 9,10-PQ induced TRPA1 channel activity without TPP in excised outsideout patches (Fig. 2D), this suggests that polyphosphates are not essential for channel activation. Nevertheless, it is evident that TPP stabilizes ( Fig. 1E and F) and potentiates ( Fig. 1H and I) 9,10-PQ-induced responses. Figure 6. C665 involvement in 9,10-PQ-induced TRPA1 channel response. Cells were superfused with 0 Ca SBS and dialyzed with Cs-aspartaterich pipette solution including 0.3 lmol/L Ca 2+ . Ramp waveform voltage pulses from À150 to +50 mV for 100 msec were applied every 5 sec. 9,10-PQ was commutatively applied to HEK-muTRPA1 cells with a C665S substitution (C665S) to examine the effects on membrane currents at À50 mV (A) and the pooled data of the peak currents evoked were summarized (B, 10 independent experiments). After application of 10 lmol/L 9,10-PQ, 30 lmol/L HC (black rectangles) was added to block TRPA1 channel current components. As a comparison, 30 lmol/L AITC was finally applied. *P < 0.05 by ANOVA. Effects of 9,10-PQ on sensitizing TRPA1 in HEK-muTRPA1 cells with C665S (C665S) (C and D) and with C621S substitutions (C621S) (E and F). The TRPA1 mutants were sensitized by Cu 2+ plus 1,10-Pher and then exposed to 9,10-PQ. As a control, each cell was exposed to 9,10-PQ only in the presence of 1,10-Pher without Cu 2+ at the beginning of each experiment. *P < 0.05 by paired Student's t-test. Bars = SEM. Using C621S and C665S mutants, we found that C621 is critical but not sufficient for channel activation by 9,10-PQ in nonsensitizing cells. In fact, TRPA1 with a C665S substitution had a substantially smaller response to 9,10-PQ (Fig. 6B), demonstrating a requirement for C665 in the activation. Since sensitization of the C665S mutant by Cu 2+ plus 1,10-Pher and higher intracellular Ca 2+ reversed the response, it is plausible that C665 may play an obligatory role in TRPA1 activation downstream of covalent modification of C621 by 9,10-PQ. In contrast, responses of the C621S mutant to 9,10-PQ were not restored by Cu 2+ plus 1,10-Pher sensitization, and were, in part, reversed by higher intracellular Ca 2+ , confirming that the high reactivity of C621 to 9,10-PQ is critical for channel activation. Consistently, it has been shown that the exceptionally high reactivity of C621 in TRPA1 contributes to channel activation by electrophiles (Bahia et al. 2016). On the other hand, double mutants with C621S and C665S substitutions sensitized by higher intracellular Ca 2+ did not significantly respond to 9,10-PQ, while two cells had a clear current activation by 10 lmol/L 9,10-PQ. The sensitized TRPA1 channel may be directly activated by 9,10-PQ via other cysteine residues and lysine residues at 620 and/or 708 (Hinman et al. 2006;Bahia et al. 2016).
The physiological significance of TRPA1 is still unclear in human non-neuronal cells, although keratinocytes, bronchial epithelial cells, and inflammatory synoviocytes express the channel (Atoyan et al. 2009;Mukhopadhyay et al. 2011;Hatano et al. 2012). In the present study, we identified that the air pollutant 9,10-PQ as an effective activator of human TRPA1. Since 9,10-PQ is included in CSE and DEP, it is likely that 9,10-PQ acutely activates TRPA1 in airway organs when inhaled. Moreover, CSE upregulates expression of TRPA1 in A549 cells (Nie et al. 2016). However, the limitation of this study is that the final concentration of 9,10-PQ around airway organs has not been determined when exposed to cigarette smoke and pollutant air. Nevertheless, a metabolite of 9,10-PQ was found in the urine of both smokers and nonsmokers, suggesting that 9,10-PQ is commonly taken into the human body (Asahi et al. 2014). Taken together, inhaled 9,10-PQ acutely and directly could activate human TRPA1, and may have some pathophysiological influence. In fact, acrolein in CSE contracted guinea pig tracheal smooth muscle via a TRPA1mediated pathway (Andre et al. 2008). In contrast, acrolein caused TRPA1-dependent tracheal relaxation in mouse (Cheah et al. 2014). Figure 7. Effects of 9,10-PQ on TRPA1 sensitization in HEK-muTRPA1 cells with C665S and C621S substitutions. TRPA1 channels were sensitized by intracellular dialysis of 3 lmol/L Ca 2+ . Cells were superfused with 0 Ca SBS. Ramp waveform voltage pulses from À150 to +50 mV for 100 ms were applied every 5 sec. 9,10-PQ was commutatively applied to HEK-wTRPA1 (WT) cells (A, four independent experiments), HEK-muTRPA1 cells with a C665S substitution (C665S) (B, eight independent experiments), and HEK-muTRPA1 cells with a C621S substitution (C621S) (C, seven independent experiments) to examine the effects on membrane currents at À50 mV, and the pooled data of the peak currents evoked were summarized (D). After application of 10 lmol/L 9,10-PQ, 30 lmol/L HC was added to block TRPA1 channel current components. As a comparison, 30 lmol/L AITC was applied. *P < 0.05 by ANOVA. Bars = SEM. In conclusion, the air pollutant 9,10-PQ can directly activate TRPA1; the activation is dependent on the channel's C621 residue, and is, in part, also regulated by the function of its C665 residue. Moreover, the quinone moiety of 9,10-PQ is essential for activation. Since 9,10-PQ is included in CSE and DEP, and is easily inhaled, 9,10-PQ may be an important irritant in airway organs. Figure 8. Effects of 9,10-PQ on TRPA1 sensitization with the double C665S and C621S mutation. TRPA1 channels were sensitized by intracellular dialysis of 3 lmol/L Ca 2+ . Cells were superfused with 0 Ca SBS. Ramp waveform voltage pulses from À150 to +50 mV for 100 msec were applied every 5 sec. (A) and (B) 9,10-PQ was commutatively applied to HEK-muTRPA1 cells with the double mutation (C621S, C665S) (A, 6-7 independent experiments) to examine the effects on membrane currents at À50 mV. Two representative current traces are shown (A). The pooled data of the peak currents evoked were summarized (B). After application of 10 lmol/L 9,10-PQ, 5 lmol/L A96 (black rectangles) was added to block TRPA1 channel current components. As a comparison, 30 lmol/L AITC was applied. (C) and (D) Time-matched control experiments superfused with the vehicle (DMSO) were performed using the double mutant. After exposure to the vehicle, 30 lmol/L AITC was applied, and vehicle-and AITC-evoked currents were summarized (D, 5 independent experiments). *P < 0.05 by paired Student's t-test. Bars = SEM. Page 13


text:
Down-regulation of PDK4 is Critical for the Switch of Carbohydrate Catabolism during Syncytialization of Human Placental Trophoblasts

Pyruvate dehydrogenase kinase (PDK) is known as a gatekeeper directing the carbon flux into glycolysis via inhibition of the pyruvate dehydrogenase complex. During syncytialization of placental trophoblasts, both ATP production and oxygen consumption are increased to meet enhanced energetic demands by syntiotrophoblasts. We hypothesized that down-regulation of PDK expression may play a central role in the switch from glycolysis to oxidative phosphorylation (OXPHOS) during syncytialization. By using primary human trophoblasts, we demonstrated that PDK4 was the dominating PDK isoform in human cytotrophoblasts, and its abundance was substantially decreased upon syncytialization, which was accompanied by decreases in lactate production and increases in ATP production. Knock-down of PDK4 expression reduced lactate production and increased ATP production, while over-expression of PDK4 increased lactate production and decreased ATP production, indicating that down-regulation of PDK4 is key to the shift from glycolysis to OXPHOS during syncytialization. Moreover, human chorionic gonadotropin (hCG)/cAMP/PKA pathway was demonstrated to be involved in the down-regulation of PDK4 expression upon syncytialization. Taken together, our findings disclosed that down-regulation of PDK4 is critical for the metabolic shift from glycolysis to OXPHOS during syncytialization, which may be a prerequisite for the proper implementation of syncytiotrophoblast functions.

of PDK2 and PDK4 expression and the consequent transition from glycolysis to OXPHOS are required for the differentiation of these cells 7 .
Of interest, human placental villous cytotrophoblast cells share many characteristics with tumor and stem cells, possessing the capability of differentiating into non-proliferative multi-nucleated syncytiotrophoblasts 8 . By covering the chorionic villi, syncytiotrophoblasts not only form the front line of nutrient exchange interface between mother and fetus but are also the major site of placental hormone synthesis. These fundamental changes in proliferative and functional properties in trophoblasts during syncytialization highly suggest that the pathway of carbohydrate catabolism may be altered in a similar way as in tumor and stem cells. Previous study has demonstrated that both ATP production and oxygen consumption are increased during syncytialization of human placental trophoblasts 9 . Given that OXPHOS consumes abundant oxygen and produces more ATP molecules, we hypothesized that the carbohydrate catabolism might shift from glycolysis to OXPHOS during syncytialization to meet the enhanced energetic demands to accomplish the sophisticated functions of the syncytiotrophoblasts, and this transition might be a consequence of down-regulation of PDK expression upon syncytialization. Here, we examined this hypothesis in an in vitro model of syncytialization of primary human placental trophoblasts 10,11 .

Results
Metabolic shift from glycolysis to OXPHOS during syncytialization. To obtain a transcriptional map of carbohydrate catabolism during syncytialization, we isolated primary human trophoblast cells from term placenta, which undergo spontaneous syncytialization under the culture condition with 10% fetal bovine serum (FBS), and then sequenced the transcriptomes of these cells before (3 hours after plating) and after (48 hours after plating) syncytialization with particular attention to carbohydrate catabolism (Fig. 1A) 10,11 . The differential expression analysis identified changes of several genes involved in carbohydrate catabolism during syncytialization ( Fig. 1B and Table S1). The Reads per Kilobases per Millionreads (RPKM) values for 5 OXPHOS-related genes (PDHE1B, DLAT, SDHB, SDHD and IDH3A) were significantly up-regulated whereas a glycolytic gene LDHB was significantly down-regulated during syncytialization (Fig. 1B and C and Table S1). These changes in gene expression were accompanied with decreases in lactate level (Fig. 1D) and increases in ATP production during syncytialization (Fig. 1E). These data suggest that a metabolic shift from glycolysis to OXPHOS occurs during syncytialization.
Down-regulation of PDK4 abundance during syncytialization. Although the transcriptomic analysis revealed that several OXPHOS-related genes were up-regulated and one glycolytic gene was down-regulated during syncytialization, the catalytic activity of the PDH complex that gates the carbon flux into OXPHOS from glycolysis is of prime importance. Since PDK inactivates PDH by phosphorylating the E1α subunit of PDH complex 1, 5 , it is logical to examine whether the alteration in PDK expression plays a critical role in the metabolic shift from glycolysis to OXPHOS during syncytialization. Transcriptomic analyses revealed that the RPKM value for PDK4 is much higher than that of any of the other three PDK family members in the trophoblasts before syncytialization ( Fig. 2A and Table S1), indicating that PDK4 is the dominant isoform of the PDK family in trophoblasts, although other members of PDK family were also detected. The RPKM values for all PDK isoforms were decreased during syncytialization, but the most dramatic reduction was observed with PDK4 ( Fig. 2A). Quantitative real time PCR (qRT-PCR) and western blotting analyses confirmed the down-regulation of PDK4 at both mRNA and protein levels during syncytialization (Fig. 2B). Consistently, immunofluorescent staining of human chorionic villous tissue obtained from early pregnancy (6-8 weeks) showed that PDK4 distributed mainly in the cytotrophoblasts which were also stained for SPINT1, a marker for cytotrophoblasts 12 , while the syncytiotrophoblasts, which were identified with staining for the β subunit of human chorionic gonadotropin (β-hCG) 10 , were stained faintly for PDK4 (Fig. 2D). Since PDK4 inhibits the activity of PDH complex by phosphorylating PDHE1α at serine 2936 , we assessed the abundance of phosphorylated E1α of PDH (pPDHE1α) at serine 293 during syncytialization. The abundance of pPDHE1α was substantially reduced in the absence of any changes in the total PDHE1α protein abundance during syncytialization (Fig. 2C). Collectively, these data suggest that PDK4 is the dominant PDK isoform expressed in placental trophoblast cells and PDK4 expression is dramatically down-regulated along with decreases in PDHE1α phosphorylation during syncytialization.
Role of PDK4 in the switch from glycolysis to OXPHOS during syncytialization. To assess whether PDK4 indeed plays a role in the metabolic switch from glycolysis to OXPHOS during syncytialization, isolated cytotrophoblasts were transfected with small interfering RNA (siRNA) targeting PDK4 or constructed plasmid expressing PDK4. Small interfering RNA-mediated knock-down of PDK4 expression significantly decreased PDHE1α phosphorylation ( Fig. 3A and B) and cellular lactate levels ( Fig. 3C) while increased cellular ATP levels (Fig. 3D). Conversely, over-expression of PDK4 increased PDHE1α phosphorylation ( Fig. 3E and F) and lactate levels ( Fig. 3G) while decreased ATP levels (Fig. 3H). These data indicate that down-regulation of PDK4 is involved in the switch of carbohydrate catabolism from glycolysis to OXPHOS during syncytialization.

Role of PDK4 in syncytiotrophoblast functions.
To examine whether manipulating PDK4 expression affects physiological functions of syncytiotrophoblasts, we measured the expression of β-hCG, a crucial hormone in pregnancy maintenance 13 , and the expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an enzyme that functions as the placental glucocorticoid barrier by inactivating biologically-active cortisol to its inactive counterpart cortisone 14 . Knock-down of PDK4 expression increased the abundance of β-hCG and 11β-HSD2 ( Fig. 4A and B), whereas over-expression of PDK4 decreased the abundance of β-hCG and 11β-HSD2 ( Fig. 4C and D). These data indicate that the shift of carbohydrate catabolism from glycolysis to OXPHOS as a result of down-regulation of PDK4 is implicated in the proper implementation of syncytiotrophoblast functions as exemplified by β-hCG and 11β-HSD2 expression in this study.
Role of hCG/cAMP/PKA pathway in the regulation of PDK4 expression during syncytialization. Among the sophisticated molecular pathways that regulate syncytialization, the cAMP/PKA pathway activated by hCG is well recognized 15,16 . In addition, the cAMP/PKA pathway is also known to be linked to carbohydrate metabolism in a number of cell types 17,18 . Thus, we postulated that activation of the cAMP/PKA pathway

Discussion
In this study, we have demonstrated that PDK4 is the dominant PDK isoform in human placental trophoblasts and its abundance is substantially decreased along with a reduction in PDHE1α phosphorylation during syncytialization. Considering that there are concurrent up-regulation of OXPHOS-related genes and down-regulation of a glycolysis-related gene as well as decreases in lactate production and increases in ATP production during syncytialization, we believe that a shift from glycolysis to OXPHOS in carbohydrate catabolism occurs in trophoblasts during syncytialization and the down-regulation of PDK4 expression plays a central role in directing carbon flux into OXPHOS from glycolysis. The crucial role of PDK4 in this switch is endorsed by reciprocal changes in the production of lactate and ATP upon siRNA-mediated knocking-down of PDK4 and over-expressing PDK4. However, it should be noted that other PDK members may also contribute to the metabolic shift since their abundance is reduced during syncytialization as well. However, we believe that PDK4 plays a dominant role in this switch because the abundance of the other PDK isozymes is much less than PDK4 and their changes during syncytialization are also much smaller than PDK4. Moreover, manipulation of PDK4 expression significantly changed the carbohydrate catabolism. Once the carbon flux is directed into OXPHOS as a result of down-regulation of PDK4, enzymes in TCA cycle which are up-regulated upon syncytialization can thus ensure efficient OXPHOS. The syncytial and cytotrophoblast layers were labeled with β-hCG (green) and SPINT1 (green) respectively. Nuclei were counterstained with DAPI (blue). n = 3; *P < 0.05; **P < 0.01; ***P < 0.001 against 3 hours; n.s., not significant.
It is known that the cAMP signaling pathway is involved in both syncytialization and carbohydrate metabolism 16,17 . Previous studies have demonstrated that activation of the cAMP pathway is a determinant of carbohydrate metabolic plasticity via up-regulation of OXPHOS-associated enzymes and enhancement of mitochondria respiration in a number of cell types 17,19 . Although PKA can directly phosphorylate a number of mitochondrial enzymes involved in OXPHOS 17,20 , PKA has been demonstrated to be incapable of phosphorylating PDHE1α 21 . This study disclosed that activation of the hCG/cAMP/PKA pathway is a causative factor for the down-regulation of PDK4 expression during syncytialization. Given the role of PDK4 versus hCG/cAMP/PKA in regulating each other, we believe that there might be a feed-forward relation between the downregulation of PDK4 expression and the accumulation of hCG production during syncytializaiton. However, at the current stage, we are unclear whether this down-regulation of PDK4 by hCG/cAMP/PKA pathway is secondary to syncytialization or a direct effect on it expression.
Accumulating evidence indicates that the status of cell differentiation is closely correlated with carbohydrate catabolic pathway and mitochondrial activity. Highly proliferative stem cells usually rely on glycolysis and have few mitochondria, while differentiating stem cells normally have an evident metabolic shift to OXPHOS 22 . Induction of carbohydrate catabolism toward glycolysis can compromise the differentiation of stem cells [22][23][24][25] . Likewise, PDK2 and PDK4, as the gatekeepers for carbohydrate catabolic pathways, have been shown to play a critical role in the transition from proliferation to differentiation of the hematopoietic stem cells 7 . Therefore, glycolysis is considered to be an indicator of cell pluripotency and poor differentiation 22 . Placental cytotrophoblast cells behave in many ways like the progenitor cells, and they are capable of differentiating into terminally differentiated syncytiotrophoblast cells 13 . Our findings that cytotrophoblasts are glycolytic are in line with their pluripotent properties, while the findings that syncytiotrophoblasts adopt OXPHOS in carbohydrate catabolism are consistent with the loss of proliferative ability. These findings endorsed a previous study showing that the mitochondrial activity is repressed in cytotrophoblasts but enhanced in syncytiotrophoblasts 26 . In addition, the  adoption of glycolysis in cytotrophoblasts can provide necessary intermediate metabolites required for proliferation. Therefore, we believe that the adoption of glycolysis in cytotrophoblasts is essential to keep their proliferative ability, while the shift from glycolysis to OXPHOS during syncytialization is not only a property of terminally differentiated syncytiotrophoblats, but can also produce adequate ATP to meet the enhanced energy demands by syncytiotrophoblasts for their sophisticated functions. Our findings that over-expression of PDK4 significantly jeopardized the expression of β-hCG and 11β-HSD2 in syncytiotrophoblasts indicate a crucial role of the switch of carbohydrate catabolism to OXPHOS in the establishment of these functions.
In conclusion, we have demonstrated in this study that PDK4 is the dominant PDK isoform in human placental trophoblasts, and down-regulation of PDK4 expression upon activation of the hCG/cAMP/PKA pathway is critical for the switch of carbohydrate catabolism from glycolysis to OXPHOS during syncytialization, which may implicate in the establishment of syncytial functions (Fig. 6).

Collection of human placental villous tissues.
Human placental villous tissues were obtained from uncomplicated early gestations (6-8 weeks) after painless induced-abortion and from term (38-40 weeks) pregnancies after elective cesarean section with written informed consents under a protocol approved by the Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. All methods were performed in accordance with the relevant guidelines and regulations. The chorionic villi from early gestation were fixed with formalin for immunochemical staining, while the fresh villous tissue from term gestation was processed for cytotrophoblast isolation.
Immunohistochemical staining. Immunohistochemical staining was performed in chorionic villous sections (n = 5) as previously described 27 . Briefly, after deparaffination and rehydration, the sections were boiled in 10 mM sodium citrate buffer for antigen retrieval and permeablized in PBS containing 0.4% Triton X-100. After blocking with goat serum and subsequent incubation with primary antibodies (Table 1), the sections were incubated with Alexa Fluor 488-or Alexa Fluor 594-labeled secondary antibodies (Proteintech, Rosemont, IL). Nuclei were stained with DAPI (1 μg/mL). The staining signals were examined under a fluorescence microscope (Zeiss, Germany).  Isolation of human cytotrophoblast cells. Cytotrophoblast cells were isolated from term human placentas using a modified Kliman's method as described previously 14 . In brief, aliquots of villous tissue were randomly removed from the maternal side of the placenta. The tissue was minced and digested with 0.125% trypsin (Sigma, St. Louis, MO) and 0.03% DNase I (Sigma) in Dulbecco Modified Eagle medium (DMEM, Gibco, Grand Island, NY). The dispersed placental cells were purified using a 5-65% Percoll (GE Healthcare Bio-Sciences, Uppsala, Sweden) gradient at step increments of 5%. After centrifugation, cytotrophoblasts were collected and cultured in DMEM containing 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% antibiotics (Gibco) at 37 °C in 5% CO 2 /95% air.

Cell treatments.
To study the roles of PDK4 in the regulation of carbohydrate metabolism during syncytialization, the cells were transfected with small interfering RNA (siRNA) targeting PDK4 (sense: GAUGCUCUGUGAUCAGUAUTT, antisense: AUACUGAUCACAGAGCAUCTT) (GenePharma Co., Ltd., Shanghai, China) or a eukaryotic vector GV230 expressing PDK4 (GeneChem Co., Ltd. Shanghai, China) immediately after isolation. Randomly scrambled siRNA or empty vector was transfected as a negative control respectively. Transfection was performed using an electroporator at 175 V for 5 milliseconds following a protocol as previously described 28  RNA sequencing. Total RNA from cultured trophoblasts before syncytialization (3 hours) and after syncytialization (48 hours) was extracted using a RNA extraction kit (OMEGA Bio-Tek, Norcross, GA) respectively.
After extraction, RNA purity and integrity were determined using a NanoDrop ® ND-2000 and an Agilent 2200 TapeStation with the following parameters: A260/A280 ratio ≥1.8, A260/A230 ratio ≥2.0 and RIN value ≥7.0. RNA-Seq libraries were prepared using a TruSeq RNA sample preparation kit (Illumina) following the manufacturer's protocol. Sequencing of the libraries was conducted on an Illumina HiSeq ™ 2500 system. A computational pipeline was used to process the RNA-seq data. Reads were aligned to human genome (UCSC hg19) using TopHat v2.1.1 with default options 29 . Differential expression analysis was performed using the Cufflinks v2.1.0 30 . Genes with RPKM ≥1 were considered expressed. The changes of PDK4 before and after syncytialization were confirmed at both mRNA and protein levels with qRT-PCR and western blotting.
Hematoxylin staining of trophoblasts. Hematoxylin staining was conducted on cultured trophoblasts before and after syncytialization. Briefly, the cells were fixed with 4% paraformaldehyde for 10 min and then washed with PBS. The cells were stained with hematoxylin (Sigma) and mounted for microscopic examination.
Quantitative real time PCR. The mRNA in extracted total RNA was reverse-transcribed to cDNA using a PrimeScript ® RT kit (TaKaRa, Dalian, China), and cDNA was applied for qRT-PCR by using power SYBR ® Premix Ex Taq TM (TaKaRa). Housekeeping gene ACTB was routinely measured for normalization. The primer sequences used in qRT-PCR are illustrated in Table 2. The relative mRNA abundance was quantified with the 2 −∆∆Ct method.
Western blotting. Whole cell lysate protein was prepared from the above treated cells by using ice-cold radio immunoprecipitation assay buffer (Active Motif, Carlsbad, CA) containing a complete protease inhibitor cocktail (Roche, Basel, Schweiz) and phosphatase inhibitor (Active Motif). A standard procedure of western blotting was performed as described previously 27 . Briefly, after determination of protein concentration with Bradford assay, 20 µg protein of each sample was electrophoresed in SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Merck Millipore). After blocking and incubation with primary antibodies (Table 1), the membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (1:5000; Proteintech). The peroxidase activities of target protein bands were detected using an enhanced chemiluminescent detection system (Merck Millipore) and visualized by using a G-Box capture system (Syngene, Cambridge, UK). The abundance of phosphorylated PDHE1α was expressed as the ratio over total PDHE1α. The abundance of PDK4 and 11β-HSD2 was expressed as the ratio over the housekeeping gene α-Tubulin.
ATP Assay. Cellular ATP abundance was determined by using an ATP Bioluminescence Assay Kit (Beyotime, Shanghai, China) following a protocol provided by the manufacturer. Briefly, the trophoblast cells (1.5 × 10 6 cells) before and after syncytialization as well as after manipulation of PDK4 expression were lysed with the lysis buffer provided in the kit. After centrifugation at 12,000 × g for 5 min at 4 °C, 20 μL of the supernatant was mixed with the detecting reagent (provided in the kit) to determine ATP concentrations with a tube-luminometer. The  protein concentration of each sample was determined with Bradford assay. The cellular ATP level was expressed as the ratio of ATP abundance over protein concentration. Lactate Assay. Cellular lactate level was measured as described previously 31 . Briefly, the trophoblast cells (1.5 × 10 6 cells) before and after syncytialization as well as after manipulation of PDK4 expression were detached from the culture plate with trypsin. After centrifugation for 10 sec at 12,000 × g, the cell pellet was collected and resuspended in 100 µL ice-cold H 2 O for 15 min. After centrifugation for 5 min at 12,000 × g, the supernatant was collected to measure lactate abundance with a Lactate Assay Kit (BioVision, Milpitas, CA). The protein concentration of each sample was determined with Bradford assay. The cellular lactate level was indicated as the ratio of lactate abundance over protein abundance.
Statistical analysis. All data are reported as mean ± SEM. The number for each experiment indicates repeated experiments using trophoblast cells from different placentas. Paired Student's t-test or one-way ANOVA test followed by the Student-Newman-Keuls test was used where appropriate to assess statistical significant differences. Significance was set at P < 0.05.
Data availability statement. All data generated or analyzed during this study are included in this published article and its Supplementary Information files.