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. 2022 Jul 13;42(7):336–342. doi: 10.1089/jir.2022.0063

Knockdown of Transmembrane Protein 150A (TMEM150A) Results in Increased Production of Multiple Cytokines

Jessica L Romanet 1, Katherine L Cupo 1, Jeffrey A Yoder 1,2,3,✉
PMCID: PMC9347386  PMID: 35834652

Abstract

Lipopolysaccharide (LPS)-induced signaling through Toll-like receptor 4 (TLR4) is mediated by the plasma membrane lipid, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and its derivatives diacylglycerol and inositol trisphosphate. Levels of PI(4,5)P2 are controlled enzymatically and fluctuate in LPS-stimulated cells. Recently, transmembrane protein 150A (TMEM150A/TM6P1/damage-regulated autophagy modulator 5) has been shown to regulate PI(4,5)P2 production at the plasma membrane by modifying the composition of the phosphatidylinositol 4-kinase enzyme complex. To determine if TMEM150A function impacts TLR4 signaling, TMEM150A was knocked down in TLR4-expressing epithelial cells and cytokine expression quantified after LPS stimulation. In general, decreased expression of TMEM150A led to increased levels of LPS-induced cytokine secretion and transcript levels. Unexpectedly, knockdown of TMEM150A in a lung epithelial cell line (H292) also led to increased cytokine levels in the unstimulated conditions suggesting TMEM150A plays an important role in cellular homeostasis. Future studies will investigate if TMEM150A plays a similar role for other TLR agonists and in other cell lineages.

Keywords: cell signaling, interleukin-8, interleukin-6, RANTES, TMEM150A, TLR4

Introduction

Transmembrane Protein 150A (TMEM150A/TM6P1/TTN1) is a member of the TMEM150/damage-regulated autophagy modulator (DRAM) family of proteins that possess 6 transmembrane domains with both termini positioned within the cytoplasm (Kerley-Hamilton and others 2007). Five members of this family have been reported in humans: DRAM1 (DRAM/FLJ11259), DRAM2 (TMEM77), TMEM150A, TMEM150B (DRAM3/TMEM224/TTN2), and TMEM150C (TTN3). DRAM1 is a target gene of tumor suppressor p53 and, along with DRAM2 and TMEM150B have been implicated in regulating autophagy (Hu and others 2019). Although a specific link between TMEM150A and autophagy has not been reported, TMEM150A transcript levels were shown to increase in the liver under fasting conditions, which are known to induce autophagy (Zhang and others 2000). In addition, TMEM150A has been shown to positively regulate phosphoinositide production at the plasma membrane suggesting a link to autophagosome formation and lysosomal fusion (Chung and others 2015; Nakamura and Yoshimori, 2017; Batrouni and others 2022).

Autophagy can be activated by infection and immune stimulation and has been linked to multiple immune-related functions (Choi and others 2018; Siqueira and others 2018). In fact, the engagement of Toll-like receptor 4 (TLR4) by the lipopolysaccharide (LPS) component of gram-negative bacteria can induce autophagy (Siqueira and others 2018). Recently it was demonstrated that DRAM1 can influence autophagy downstream of TLR4/MyD88 signaling in a prosurvival response to infection (van der Vaart and others 2014).

Based on the observations that multiple TMEM150/DRAM family members have been implicated in autophagy and that, through its interactions with phosphatidylinositol 4-kinase (PI4KIIIα) TMEM150A can regulate the production of PI(4,5)P2 (a component in the TLR4 pathway), we hypothesized that TMEM150A plays a role in regulating TLR4 signaling. As no previous studies have addressed the role of TMEM150 in innate immune signaling pathways, we investigated how knockdown of TMEM150A would impact TLR4-induced cytokine production and observed that disruption of TMEM150A led to significant increases in TLR4-induced cytokine secretion. Parallel increases in cytokine transcript levels suggest that TMEM150A may be regulating cytokine production rather than cytokine release. Unexpectedly, we found that knockdown of TMEM150A without TLR4 activation also led to increased cytokine production in a lung epithelial cell line (H292), suggesting that TMEM150A is essential for maintaining cytokine homeostasis. In summary, we provide the first evidence that TMEM150A plays a role in regulating cytokine production.

Materials and Methods

Cell culture

HEK293 cells stably expressing human TLR4, MD2, and CD14 (293/TLR4-MD2-CD14, hereafter referred to as HEKTLR4; Invivogen) and NCI-H292 cells (hereafter referred to as H292; ATCC CRL-1848) were cultured according to suppliers' instructions. The authenticity of cell lines was verified using STR markers and found to be free of mycoplasma (IDEXX BioAnalytics).

siRNA transfection

Three TMEM150A siRNA (“A,” “B,” and “C”) and one scrambled control siRNA were obtained from Origene Technologies (SR315062 and SR30004): TMEM150A siRNA “C” was most effective and used for these studies. siRNA (5 pmol) was transfected into cultured cells using Lipofectamine RNAiMax (Life Tech 13778) according to the manufacturer's protocol. HEKTLR4 cells were transfected after the cells were adherent postplating (∼18 h). H292 cells were transfected concurrent with plating. TMEM150A knockdown was validated by reverse transcriptase (RT)–quantitative polymerase chain reaction (qPCR) analyses.

Western blots

Cells were lysed with concentrated radioimmunoprecipitation assay buffer (0.2% sodium dodecyl sulfate, 1% sodium deoxycholate, 2% NP-40, 10 mM sodium pyrophosphate, and 100 mM sodium fluoride) containing Halt™ Protease Inhibitor Cocktail (78430; ThermoFisher). Lysates were agitated on ice for 15 min, sonicated, agitated for an additional 10 min, and centrifuged at 14,000 × g for 15 min at 4°C. Supernatants were collected and stored at −80°C.

Protein concentrations were determined (BCA Protein Assay, Pierce, Thermo) and equal amounts of protein were loaded onto 4%–20% gradient or 10% Mini-PROTEAN TGX Stain-free Gels (BioRad) for electrophoresis. After electrophoresis, gels were activated for 1 min with UV per manufacturer's instructions and proteins were transferred to PVDF membranes (Transblot Turbo Transfer system; BioRad). Membranes were blocked in Tris-buffered-saline and 0.1% Tween (TBST) with 5% skim milk (TBSTM) for 1 h at room temperature. Membranes were incubated with anti-TMEM150A antibody (NBP1-81885; Novus Biologicals) overnight at 4°C at a dilution of 1:750 or with anti-β-actin antibody (Cell Signal) for 1 h at room temperature at a dilution of 1:5,000 in TBSTM. Membranes were washed with TBST and then incubated with horseradish peroxidase-conjugated antibodies in TBSTM for 1 h at room temperature. Membranes were washed and developed using BioRad Clarity ECL and visualized with a ChemiDoc MP system (BioRad). Image normalization and quantification was completed with BioRad Image Lab software.

Enzyme-linked immunosorbent assay/Milliplex

HEKTLR4 or H292 cells were transfected with siRNA or left untreated. One day after transfection, media were changed and cells were stimulated with 0, 30, 100, or 300 ng/mL LPS (Escherichia coli 055:B5 Sigma L2880) for 18 h. At 18 h, cell supernatants were collected, centrifuged for 5 min at 4°C, aliquoted, and stored at −80°C. Adherent cells were washed with phosphate-buffered saline, trypsinized, and counted. Supernatant was evaluated with enzyme-linked immunosorbent assays (ELISAs) to measure protein secretion of CXCL8 (IL8), Rantes (CCL5), and IL6 (R&D Systems; Quantikine D8000C, DRN00B, D6050). Readouts were analyzed by 4-parameter logistic regression and output was normalized to cell counts. For Milliplex analysis, cells and supernatant were treated as described previously and protein secretion measured by a Milliplex Map Human Cytokine Immunoassay (Millipore Catalog No. HSCYMAG60SPMX13).

RNA isolation and quantitative real-time PCR

Cells were homogenized (Qiagen; QIAshredder) and RNA was isolated (Qiagen; RNeasy Mini Kit). RNA quantity and quality was verified with a NanoDrop and an Agilent 2100 Bioanalyzer. RNA samples with integrity numbers <8.0 were excluded from analyses. cDNA was synthesized using equal quantities of RNA and the Maxima First-Strand cDNA Synthesis Kit (Thermo K1671). Quantitative real-time PCR was performed using Applied Biosystems Taqman Universal PCR Master Mix II and Taqman Gene Expression array (4413266), glyceraldehyde-3-phosphate dehydrogenase (Hs02758991_g1), IL10 (Hs00961622_m1), IL12A (Hs01073447_m1), IL6 (Hs00985639_m1), CXCL8 (Hs00174103_m1), CCL5 (Hs00982282_m1), and tumor necrosis factor (TNF) (Hs01113624_g1). Fold changes in transcript levels were calculated using the 2−ΔΔCt method.

Results

Knockdown of TMEM150A increases TLR4-mediated CXCL8 production

To investigate a possible link between TLR-mediated immune response and TMEM150A function, TMEM150A expression was knocked down in a cell line with a defined TLR signaling pathway. Specifically, a modified version of the human embryonic kidney cell line, HEK293, which stably expresses TLR4, and 2 cofactors necessary for LPS response—MD2 and CD14 (hereafter referred to as HEKTLR4) were used. When exposed to LPS, this cell line is activated solely through the TLR4 pathway as other TLRs, including TLR2, are either not present or nonfunctional (Takeuchi and others 1999).

By combining siRNA techniques to knockdown TMEM150A in HEKTLR4 cells (Fig. 1A) and challenging them with different concentrations of LPS, we assessed how loss of TMEM150A affects CXCL8 release after TLR4 stimulation (Fig. 1B)—CXCL8 is the only cytokine effectively produced by HEKTLR4 cells. We observed that, after exposure to 30, 100, and 300 ng/mL LPS, TMEM150A-deficient HEKTLR4 cells secreted more CXCL8 protein than control cells. Although this increase in CXCL8 secretion reflects an increase in the cellular release of CXCL8 stores, it might also reflect increases in CXCL8 transcript and protein production.

FIG. 1.

FIG. 1.

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Knockdown of TMEM150A increases TLR4-mediated CXCL8 production. (A) Endogenous TMEM150A protein levels are effectively knocked down by TMEM150A siRNA. HEKTLR4 cells were transfected with TMEM150A siRNA or control siRNA or not transfected and then stimulated with 0, 30, 100, or 300 ng/mL of LPS for 18 h. Knockdown of protein was evaluated by Western blot with an anti-TMEM150A antibody. Uncropped Western blot images are provided in Supplementary Fig. S1. (B) HEKTLR4 cells were transfected with TMEM150A siRNA, control siRNA, or not transfected and then stimulated with 0, 30, 100, or 300 ng/mL of LPS for 18 h and cell culture supernatant collected. Levels of CXCL8 protein in supernatants were quantified by Milliplex technology (left). Fold change was calculated based on protein levels in nontransfected, 0 ng/mL LPS-treated cell supernatant. Results were confirmed by ELISA (right). ELISA data are representative of 3 biological replicates. (C) HEKTLR4 cells were treated as described in (A) and 18 h after LPS exposure, RNA was extracted from cells and CXCL8 transcript levels measured by qPCR. Relative CXCL8 levels were normalized to levels of β-actin and fold-change calculated by comparison with nontransfected 0 ng/mL LPS samples. PCR data are representative of 2 biological replicates. ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; PCR, polymerase chain reaction; TLR4, Toll-like receptor 4.

To investigate if loss of TMEM150A affects transcription of CXCL8, qPCR was used with HEKTLR4 cells in which TMEM150A was knocked down. Increases in CXCL8 transcript levels were observed in TMEM150A knockdown cells after LPS stimulation (Fig. 1C). These data implicate TMEM150A in regulating CXCL8 production at the transcript level. Of importance, ELISA-based experiments using HEK293 cells that do not stably express TLR4 failed to detect any CXCL8 production after LPS exposure (not shown). In this experimental cell model, activation of TLR4 in the absence of TMEM150A leads to significantly increased amounts of CXCL8 compared with control cells. The observed increase in LPS-induced CXCL8 protein release and CXCL8 transcript levels in TMEM150A-deficient cells indicate that TMEM150A plays a role in regulating the TLR4-mediated induction of CXCL8 production.

Knockdown of TMEM150A in human lung epithelial cells causes increased protein and transcript levels of multiple cytokines

To determine if the changes in cytokine levels after knockdown of TMEM150A were limited to HEKTLR4 cells and/or limited to CXCL8 production, we evaluated if knockdown of TMEM150A would have a more global response and affect additional cytokines in a lung carcinoma epithelial cell line, NCI-H292s (H292). Because H292 cells originate from barrier-type epithelium at the interface of the interstitium and vasculature as well as the outside air and inhalants, H292s are more endogenously equipped to respond to immune stimuli in comparison with HEK cells. Because the epithelium of the lung is an area of trafficking vital nutrients and microbes, and is thus in constant contact with potential pathogens, H292 lung epithelial cells act as a model for this important barrier where cytokine production and control are critical and are known to secrete cytokines like CXCL8, CCL5 (Rantes), and IL6 (Kato and Schleimer, 2007).

Additional cytokine transcripts, including IL7, IL10, IL12B, interferon gamma (IFN-γ), and TNF have been reported in airway epithelial cells (Matsukura and others 1996; Adachi and others 1997; Kato and Schleimer, 2007; Lee and others 2010; Lam and others 2011; Oyanagi and others 2017). Therefore, we applied our protocol for siRNA knockdown of TMEM150A and LPS exposure using H292 cells to investigate a potential role for TMEM150A in regulating a broader cytokine response (Matsukura and others 1996; Adachi and others 1997; Lam and others 2011).

We quantified secretion of CXCL8, IL6, and CCL5 after LPS stimulation by ELISA (Fig. 2A), and demonstrated that IL6 and CCL5 protein production was significantly higher after LPS exposure in cells lacking TMEM150A as compared with nontransfected and scrambled siRNA control cells. CXCL8 showed the same trend in H292 cells as observed for HEK cells, although with a more variable response. To investigate if TMEM150A knockdown alters secretion levels of additional cytokines, Milliplex map technology was used. Figure 2B provides the average cytokine fold change for IL7, IL10, IL12B (which dimerizes with IL12A to produce IL12), IFN-γ, and TNF and recapitulates the trend observed by ELISA for CXCL8, IL6 and CCL5. Of interest, increased levels of these cytokines were observed with the knockdown of TMEM150A without LPS exposure, and exposure to LPS further increased cytokine levels. In contrast, granulocyte macrophage–colony stimulating factor production increased only after exposure to 100 and 300 ng/mL of LPS.

FIG. 2.

FIG. 2.

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TMEM150A knockdown affects cytokine regulation in human mucoepidermoid pulmonary carcinoma cell line (H292). TMEM150A knockdown results in increased protein production of multiple cytokines. H292 cells were not transfected, or transfected with control siRNA or TMEM150A siRNA. Cells were stimulated with 0, 30, 100, or 300 ng/mL of LPS. Supernatant was collected after 18 h of LPS stimulation (48 h post-siRNA transfection), and cells were counted. (A) Cytokine protein production was quantified by ELISA and normalized to cell counts. Fold change was calculated using supernatants from not transfected, 0 ng/mL LPS exposed cells as baseline. ELISA data presented are the average (±SEM) of 3 biological replicates. Statistical analyses were performed by 2-factor ANOVA testing significance and interaction of siRNA and LPS. Note that TMEM150A siRNA-treated samples, independent of LPS exposure, produced significantly more cytokines than controls (**P < 0.001, *P < 0.05). (B) H292 cells were treated as described previously and supernatant was quantified by Milliplex map technology for multiple cytokines. Values represent the average of 2 biological replicates. (C) Cytokine transcript levels were quantified by qPCR. H292 cells were treated as described previously and RNA extracted 18 h after LPS stimulation (48 h post-siRNA transfection). Relative transcript levels were normalized to levels of GAPDH and fold-change calculated by comparison with nontransfected 0 ng/mL LPS samples. Data presented are the average (±SEM) of 3 biological replicates. Statistical analyses were performed by 2-way ANOVA. With the exception of IL12A, TMEM150A siRNA-treated samples displayed significantly higher cytokine transcript levels than all controls (**P < 0.001, *P < 0.05). IL12A transcript levels were increased by both TMEM150A siRNA and the control siRNA. ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

To investigate if these cytokine aberrations were the result of changes in transcript levels, we used a multi-array qPCR strategy (Fig. 2C). At the transcript level, CCL5, IL6, CXCL8, and TNF showed similar expression profiles and reflected our findings of changes in protein levels. TMEM150A-deficient H292 cells displayed an increase in transcripts of these cytokines compared with controls. No and low (30 ng/mL) levels of LPS stimulation elicited the highest transcript fold changes versus 100 ng/mL LPS. IL12A transcripts did not mirror IL12B protein secretion changes. IL12A transcript levels of both TMEM150A-deficient cells and siRNA scrambled control cells were similar (Fig. 2C). Overall, reduction of TMEM150A leads to an increase in both transcript and protein levels of multiple cytokines suggesting that TMEM150A plays a role upstream of cytokine transcription.

Discussion

It was recently reported that PI4KIIIα, the enzyme that produces PI(4)P, which can then be phosphorylated to yield PI(4,5)P2, is thought to exist in 2 distinct protein complexes within the plasma membrane (Batrouni and others 2022). Complex I, which is present in liquid ordered-like domains of the membrane and does not include TMEM150A, reflects the initial recruitment of PI4KIIIα to the membrane via EFR3B. Complex II, which is present in liquid disordered-like domains of the membrane and includes TMEM150A, is more efficient for PI(4,5)P2 synthesis. In the model for complex II, TMEM150A associates directly with both PI4KIIIα and EFR3B (Batrouni and others 2022).

It has been suggested that changes in EFR3B palmitoylation regulates its interactions with TMEM150A and the relocation of PI4KIIIα between different membrane domains and this relocation of PI4KIIIα relies on TMEM150A. Although speculative, we predict that knockdown of TMEM150A results in less complex II and an imbalance of where PI(4,5)P2 is produced within the membrane (liquid ordered-like versus liquid disordered-like domains) and/or an overall decrease in PI(4)P and subsequently PI(4,5)P2.

If knockdown of TMEM150A leads to a decreased or altered distribution of PI(4,5)P2 in the plasma membrane, this could significantly alter the signaling capacity of cells. First, TIR domain-containing adaptor protein (TIRAP), which is an essential component of TLR4 (and TLR2) signaling, associates specifically with PI(4,5)P2-enriched membrane domains (Le and others 2014; Zhao and others 2017). It is possible that a reduction in PI(4,5)P2 in the plasma membrane could alter the availability of TIRAP and thus the signaling potential of TLR4. Second, altered PI(4,5)P2 production could limit or reduce the signaling capacity of phospholipase C (PLC) enzymes that cleave PI(4,5)P2 into second messengers, inositol trisphosphate and derivatives diacylglycerol, which mediate an extensive number of cellular functions (Kudo and others 2016; Zhu and others 2018). At least one PLC, PLC-δ1, normally inhibits LPS-induced cytokine expression (Ichinohe and others 2007), so limiting its function could lead to increased cytokine production.

Our results are consistent with 2 mechanisms for increasing cytokine production—the first would be through LPS stimulation of TLR4 and the second would be specific to the role of TMEM150A in the plasma membrane. Whether our results reflect that knockdown of TMEM150A leads to dysregulated PI(4,5)P2 levels and altered TLR and PLC functions remains to be determined.

Finally, it is worth noting that TMEM150A is not exclusively located in the plasma membrane. Overexpression of a TMEM150A-GFP fusion protein in a liver cell line resulted in fluorescence that suggested TMEM150A protein localizes “around the nuclei” and in the plasma membrane (Zhang and others 2000). Similarly, overexpression of a different TMEM150A-GFP fusion protein in HeLa cells resulted in fluorescence primarily at the plasma membrane but under certain conditions, also in a punctate pattern throughout the cell (Chung and others 2015). Furthermore, a human interactome report (Bioplex Network) that utilized high-throughput affinity-purification mass spectrometry, identified TMEM150A as colocalizing with nuclear proteins (Huttlin and others 2015).

Accordingly, TMEM150A may interact with PI4KIIIα in and around the nucleus to regulate or sequester its activity. Another possibility is that the interaction of TMEM150A and PI4KIIIα could modify cytoskeleton arrangements, as it has been reported that IL6 and CXCL8 mRNA stability is affected by cytoskeleton distortion in airway epithelial cells (van den Berg and others 2006). Alternatively, TMEM150A's interactions with PI4KIIIα could affect autophagic flux and thereby cytokine production. There is evidence that PI(4)P, the direct product of PI4KIIIα activity, is central to late phagolysosomal maturation (Jeschke and others 2015) and hence is significant in moderating autophagic flux. Deeper investigation into the interactions between TMEM150A and PI4KIIIα, with a focus on how they affect autophagic flux, may be the next step in understanding this somewhat universal inflammatory upregulation in TMEM150A-deficient cells.

Summary

This report is the first to identify TMEM150A as an important mediator in regulating the production of multiple cytokines. Despite the consistent observed increases in cytokine protein secretion after TMEM150A knockdown and LPS stimulation, changes in cytokine levels vary depending on the concentration and timing of LPS exposure as well as the cell type. Because reported transcript levels are from a single time point (18 h) after LPS stimulation, they may reflect a feedback mechanism to dampen the immune response. As these experiments were limited to epithelial cell lines, it will be of interest to determine if knockdown of TMEM150A has a similar effect on other cell types. Similarly, it will be important to determine if other members of this family (eg, TMEM150B and TMEM150C) play a similar role in cytokine production.

Nevertheless, these data suggest TMEM150A plays an important role in regulating cytokine production outside the TLR4 pathway in epithelial cells, and may play a more global regulatory role in other cell types. This model is in line with its relationship to both PI4KIIIα and DRAM family members that modulate autophagy, which are connected to, but not isolated from, the TLR4 immune response. More research is needed to decipher the mechanism by which TMEM150A modulates cytokine production.

Supplementary Material

Supplemental data
Supp_FigS1.pdf (298.1KB, pdf)

Acknowledgments

The authors thank Gretchen Scheffe and Anne Crews for technical assistance and Shila Nordone and Phil Sannes (NC State University) for cell lines. Biomarker profiling was performed under the management of Dr. Heather E. Lynch and direction of Dr. Gregory D. Sempowski in the Immunology Unit of the Duke Regional Biocontainment Laboratory.

Authors' Contributions

J.L.R.: conceptualization, methodology, investigation, writing—original draft preparation. K.L.C.: investigation, writing—reviewing and editing. J.A.Y.: conceptualization, visualization, supervision, writing—reviewing and editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research was supported by funding from the United States National Institutes of Health [R21 AI076829 (J.A.Y.) and T32 GM008776 (J.L.R.)] and from the North Carolina State University College of Veterinary Medicine (J.A.Y.). Biomarker profiling was performed under the management of Dr. Heather E. Lynch and direction of Dr. Gregory D. Sempowski in the Immunology Unit of the Duke Regional Biocontainment Laboratory that received partial support for construction from NIH, Grant No. UC6-AI058607. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.

Supplementary Material

Supplementary Figure S1

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Associated Data

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Supplementary Materials

Supplemental data
Supp_FigS1.pdf (298.1KB, pdf)

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