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Occludin is a target of Src kinase and promotes lipid secretion by binding to BTN1a1 and XOR

['Yunzhe Lu', 'School Of Life Science', 'Technology', 'Shanghaitech University', 'Shanghai', 'Tao Zhou', 'University Of Chinese Academy Of Sciences', 'Beijing', 'Chongshen Xu', 'Rui Wang']

Date: 2022-01 

Lipid droplets (LDs) have increasingly been recognized as an essential organelle for eukaryotes. Although the biochemistry of lipid synthesis and degradation is well characterized, the regulation of LD dynamics, including its formation, maintenance, and secretion, is poorly understood. Here, we report that mice lacking Occludin (Ocln) show defective lipid metabolism. We show that LDs were larger than normal along its biogenesis and secretion pathway in Ocln null mammary cells. This defect in LD size control did not result from abnormal lipid synthesis or degradation; rather, it was because of secretion failure during the lactation stage. We found that OCLN was located on the LD membrane and was bound to essential regulators of lipid secretion, including BTN1a1 and XOR, in a C-terminus–dependent manner. Finally, OCLN was a phosphorylation target of Src kinase, whose loss causes lactation failure. Together, we demonstrate that Ocln is a downstream target of Src kinase and promotes LD secretion by binding to BTN1a1 and XOR.

Funding: This work was supported by grants from the Ministry of Science and Technology of China (2017YFA0103502 to P.L.) and the NSF of China (31671494 to P.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Lu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

As a founding member of tight junctions, Ocln was initially considered to be essential for tight junctions’ integrity and function [ 22 , 23 ]. However, recent studies show that Ocln regulates an array of developmental processes, including stem cell biology in the developing brain, which are not directly related to tight junctions’ functions [ 24 , 25 ]. Likewise, we recently found that Ocln regulates milk protein secretion via SNARE-dependent exocytosis [ 26 ]. Here, we describe an additional phenotype in Ocln null mammary glands in which LD secretion is defective.

As a major secretory organ, the mammary gland is an ideal model for understanding the mechanism that regulates lipid secretion [ 11 – 15 ]. Loss-of-function studies in mice support the involvement of several genes, including Th-POK, Cidea, TDP-23, Btn1a1, Xor, and Src kinase in lipid secretion regulation. Among them, Cidea and TDP-23 regulate lipid secretion via modulating Btn1a1 and Xor mRNA expression, transcriptionally and posttranscriptionally, respectively [ 16 , 17 ]. By contrast, Th-POK indirectly affects LD secretion by transcriptionally regulating lipid synthesis [ 18 ]. So far, only Btn1a1 and Xor are thought to directly participate in the interactions between LDs and the plasma membrane and the secretion event [ 19 , 20 ]. However, the mechanism by which Btn1a1 and Xor regulate lipid secretion, especially how they facilitate the LD extrusion process and what additional partners they might require, has remained unclear [ 5 – 7 ]. Likewise, how Src kinase regulates lipid secretion has also remained elusive at present [ 21 ].

Lipids may be secreted via one of 2 independent pathways. In both hepatocytes and enterocytes, for example, lipids in the form TGs are incorporated into lipoproteins, which then take the conventional protein secretion route and are secreted via a SNARE-dependent membrane fusion event between secretory vesicles and the plasma membrane [ 10 ]. In mammary epithelial cells (MECs), however, LDs are secreted via budding, i.e., mature LDs at the apical domain of MECs are enveloped by the plasma membrane, pinch off from MECs through an unknown mechanism, and enter the alveolar lumen. With the addition of lipid bilayer from the plasma membrane, secreted LDs now have 3 layers of phospholipids. They are sometimes called milk fat globules (MFGs) to differentiate them from intracellular LDs [ 5 – 7 ].

An LD may grow via increased lipid synthesis or, to a lesser extent, the fusion between 2 existing LDs [ 8 ]. Because there are in the cytoplasm multiple forms of lipases, or TG degradation enzymes, for an LD to grow, it needs to be shielded by coat proteins such as Perilipin-2 (PLIN2, also known as ADRP) from these cytoplasmic lipases [ 1 , 9 ]. Thus, an LD grows if the local synthesis of lipids, catalyzed by enzymes on LD membranes, outpaces degradation. Therefore, an important mechanism of LD size control is by regulating the number of shield proteins such as PLIN2 on the LD surface. At the maintenance stage, lipid synthesis reaches an equilibrium with degradation.

Contrary to their relatively simple molecular composition, LDs have a complex life cycle comprising formation, growth, and maintenance stages [ 4 ]. In certain cell types, including hepatocytes and mammary gland cells, LDs also undergo maturation and secretion stages [ 5 – 7 ]. Briefly, LD formation starts in the endoplasmic reticulum (ER), where the accumulation of TGs in the shape of a lens forms in the cytoplasmic layer of the ER membrane. Through a poorly understood mechanism, these micro-LDs then separate from the ER and enter the growth stage, in which LDs grow [ 1 , 4 ].

Lipid droplets (LDs) are microscale structures that are ubiquitous in almost all eukaryotic cells. They are composed of an inner hydrophobic core of neutral storage fats, the triglycerides (TGs), or cholesterol esters, and an outer layer of protein-coated phospholipid monolayer membrane [ 1 ]. An important means that a cell regulates its energy storage is by controlling LD sizes, which can vary from less than tens of nanometers in diameter in most cells to hundreds of micrometers that can fill an entire adipocyte [ 2 ]. Although initially considered as passive energy storage depots, LDs have gained increasing recognition as a new organelle essential for various fundamental cellular processes, including lipid trafficking, vesicular transport, and metabolism [ 1 , 3 ].

Results

Normal mRNA expression of genes regulating triacylglycerol synthesis and degradation in Ocln null cells The increase in LD size in Ocln null alveoli at the L2 stage could result from an increase in TG synthesis or a decrease in its degradation in the mutant gland. We, therefore, examined the mRNA expression of the critical enzymes during TG synthesis and degradation (Fig 3A) [1]. In our previous study, we used single-cell RNA sequencing (scRNA-seq) technology and examined the transcriptomics of both control MECs and Ocln null MECs at the L2 stage [26]. Therefore, we mined the same scRNA-seq datasets and compared the mRNA expression of the critical lipid metabolic genes. We found that the mRNA expression of TG synthesis genes, including Acsl1, Acss1, Acss2, Gk5, etc., was similar in individual luminal cells of Ocln null and control mammary glands at the L2 stage (Figs 3B and S2A). Likewise, the mRNA expression of TG degradation genes, including Lipe, Dgka, Agk, Lipa, etc., was also similar between Ocln null and control mammary glands at this stage (Figs 3B and S2A). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Triacylglycerol synthesis and degradation are relatively normal in Ocln null cells. (A) Overview diagram of de novo TG synthesis and the enzymes involved. (B) Relative gene expression levels of some of the genes in the TG synthesis pathway based on data from the scRNA-seq dataset (see Materials and methods). Each dot indicates the expression level based on log2 of the genes in a single cell. (C) Levels of mRNA expression as detected by qPCR of several key TG synthesis genes at the 10-wk, P5, P12, P17, and L2 stages. Values were normalized against actin expression, and gene expression at 10 wk of age was set as the base value against which other stages were compared. Graph shows mean ± SD. (D) Levels of Plin2 mRNA expression as detected by qPCR at the 10-wk, P5, P12, P17, and L2 stages. The number of female mice at each stage used were: Ocln−/+ (n = 3) and Ocln−/− (n = 3). (E) Immunoprecipitation assay to determine protein binding between OCLN and PLIN2. OCLN was tagged by Flag protein, whereas PLIN2 was tagged by HA. Antibody against Flag was used for immunoprecipitation, and antibody against HA was used for subsequent western blotting analysis. No binding of PLIN2 to OCLN was detected in this assay. Note that the individual numerical values that underlie the summary data here are listed in S1 Data file. HA, hemagglutinin; KD, kilodalton; L, lactation; Ocln, Occludin; P, pregnancy; PLIN2, Perilipin-2; qPCR, quantitative PCR; scRNA-seq, single-cell RNA sequencing; TG, triglyceride. https://doi.org/10.1371/journal.pbio.3001518.g003 Next, we sought to validate the results from the scRNA-seq data using quantitative PCR (qPCR). To identify a suitable housekeeping gene as an internal control for the qPCR analysis, we first examined Actb and Gapdh mRNA expression in our scRNA-seq dataset. We found that Actb and Gapdh mRNA expression was similar in both Ocln null and control mammary glands at the L2 stage (S3A Fig). Furthermore, when either Actb or Gapdh was used as an internal control for each other in qPCR assays, mRNA expression of the other gene was similar between Ocln null and control mammary glands. Likewise, Cldn10, whose expression was not affected by Ocln loss [26], was also found to have similar levels of mRNA expression irrespective of whether Actb or Gapdh was used as an internal control for qPCR (S3B Fig). These data suggest that both Actb and Gapdh were a good choice for internal controls for qPCR assays. Therefore, we chose Actb as an internal control for our qPCR validation assays. We found that the mRNA expression of both TG synthesis and degradation genes were similar between Ocln null and control MECs at various developmental stages, including the 10-wk, P5, P12, P17, and L2 stages (Figs 3C and S2B). A possibility exists where Ocln may affect the expression or function of Plin2, which coats the LDs and protects them from cytoplasmic lipases and thus lipolysis [1,9]. To address this possibility, we first analyzed mRNA expression of Plin2 at several developmental stages, including the 10-wk, P5, P12, P17, and L2 stages. We found that Plin2 mRNA expression was similar between Ocln null and control MECs at these stages (Fig 3D). Next, we sought to determine whether OCLN could interfere with PLIN2 function by binding to it. We “tagged” both OCLN and PLIN2 at their N-termini using FLAG and hemagglutinin (HA) peptides, respectively. The fusion proteins were then subjected to co-immunoprecipitation (co-IP) assays to test their potential binding. We found that OCLN did not bind to PLIN2 in this assay (Figs 3E and S4A). Finally, we determined whether OCLN and PLIN2 could colocalize in the living cells, which could also show their potential binding or interactions. Thus, we fused OCLN and PLIN2 proteins at their N-termini with a green fluorescent protein (GFP) and a mCherry protein, respectively, which were then introduced into primary MECs using lentiviral vectors. Using fluorescence confocal microscopy, we found OCLN did not colocalize with PLIN2 protein (S4B Fig; S1 Table, S1 Movie). Taken together, the data suggest that the increase in LD size in Ocln null alveoli at the L2 stage unlikely results from an increase in TG synthesis or a decrease in TG degradation in the mutant gland.

OCLN localizes to membranes of lipid droplets and milk fat globules OCLN may regulate LD secretion directly or indirectly. If OCLN is present at one or more places of the LD biosynthesis and secretion route, then the likelihood that OCLN plays a direct role in lipid secretion is greater than otherwise when it is absent. Using immunofluorescence, we found that OCLN was indeed present on MFGs in the milk (Fig 5A–5C). When ultrathin mammary gland tissue sections from the L2 stage were examined using immuno-EM, we found that OCLN, as shown by gold particles, was on the LD surfaces (Fig 5D–5D2”). Moreover, OCLN was also present at the contact sites between the mature LD, as judged by both its diameter and apical localization, and the plasma membrane (Fig 5E and 5E’). PPT PowerPoint slide

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TIFF original image Download: Fig 5. OCLN localizes to membranes of lipid droplets and milk fat globules. (A-C) Immunofluorescence of OCLN protein (red, A) on milk fat droplets, revealed by Bodipy (green, B) and overlay (C). Scale bar, 5 μm. Samples were taken from female mice at the L3 stage. A total of 659 LDs were examined. (D-E’) Localization of OCLN protein in luminal epithelial cells as detected by immunoelectron microscopy at the L2 stage. Primary antibodies against OCLN were visualized by a secondary antibody coupled to gold particles (black arrowheads). Areas in red or green-dotted boxes indicate close-up views. (D’, D1”-D2”) are progressive close-up views of LDs in (D) and (D’), respectively. (E, E’) indicate a close juxtaposition of an LD and apical plasma membrane. Scale bars are as indicated. ER, endoplasmic reticulum; LD, lipid droplet; Lu, lumen; Nu, nucleus. https://doi.org/10.1371/journal.pbio.3001518.g005 Together, the data indicate that OCLN is present along the routes of LD development and secretion, suggesting that it plays a direct role in LD metabolism.

OCLN binds to and colocalizes with lipid secretion regulators BTN1 and XOR BTN1A1 and XOR are essential regulators of LD secretion that are thought to be directly involved in the juxtaposition of LD and the plasma membrane [5,6]. Therefore, we decided to examine the possibility that OCLN may participate in LD secretion by regulating Btn1a1 and Xor function. To this end, we first determined whether Ocln might regulate mRNA expression of Btn1a1 and Xor, which, using qPCR, we found that the mRNA expression of neither Btn1a1 nor Xor was affected by Ocln loss at several stages of mammary gland development, including the 10-wk, P5, P12, P17, and L2 stages (Fig 6A and 6B). These data thus suggest that the cause of LD secretion failure was not due to abnormal expression of Btn1a1 and Xor. PPT PowerPoint slide

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TIFF original image Download: Fig 6. OCLN binds to and colocalizes with lipid secretion regulators BTN1a1 and XOR. (A, B) Levels of mRNA expression as detected by qPCR of LD secretion regulators Btn1a1 (A) and Xor (B) in mammary gland epithelial cells at the 10-wk, P5, P12, P17, and L2 stages. Values were normalized against actin expression, and gene expression at 10 wk of age was set as the base value against which other stages were compared. Graph shows mean ± SD. The number of female mice at each stage used were: Ocln−/+ (n = 3) and Ocln−/− (n = 3). (C, D) Protein binding between OCLN and BTN1a1 (C) and between OCLN and XOR (D) as detected by co-IP assays. OCLN was tagged by Flag protein, whereas BTN1a1 and XOR were tagged by HA. Antibody against Flag was used for immunoprecipitation, and antibody against HA was used for subsequent western blotting analysis. (E, F) Time course of localization of OCLN and BTN1a1 (E) or XOR (F) as detected by fluorescent microscopy. GFP was fused in-frame with OCLN at the N-terminus, whereas mCherry was fused in-frame with BTN1a1 (E) or XOR (F). White arrowheads mark OCLN and BTN1a1 (E) and XOR (F) particles over the time course of observation. Note that 54% and 44% of OCLN particles (S1 Table) colocalized with BTN1a1 (E) and XOR (F) particles, respectively. A total of 19 GFP-OCLN and mCherry-BTN1a1 double-positive cells and 28 GFP-OCLN and mCherry-XOR double-positive cells were examined in this experiment. (G, H) Protein binding between OCLN truncations and BTN1a1 (G) and between OCLN truncations and XOR (H) as detected by co-IP assays. OCLN truncations were tagged by Flag protein, whereas BTN1a1 and XOR were tagged by HA. Antibody against Flag was used for immunoprecipitation, and antibody against HA was used for subsequent western blotting analysis. Note that the individual numerical values that underlie the summary data here are listed in S1 Data file. co-IP, co-immunoprecipitation; GFP, green fluorescent protein; HA, hemagglutinin; KD, kilodalton; L, lactation; LD, lipid droplet; P, pregnancy; qPCR, quantitative PCR. https://doi.org/10.1371/journal.pbio.3001518.g006 Alternatively, OCLN may bind to BTN1a1 and XOR to form a multiplayer complex, which is then required for LD secretion. To test whether OCLN is bound to BTN1a1 and XOR, we “tagged” OCLN and BTN1a1 or XOR proteins at their N termini with FLAG and HA peptides, respectively. Tagged proteins were then subjected to co-IP to test their potential binding. We found that OCLN bound to both BTN1a1 and XOR in this assay (Fig 6C and 6D). These in vitro data thus show that OCLN could bind to BTN1a1 and XOR. Next, we sought to examine whether OCLN could bind to BTN1a1 and XOR proteins in vivo using the above colocalization assay. To this end, we fused OCLN and BTN1a1 or XOR proteins at their N-termini with mCherry and GFP, respectively. Lentiviral constructs expressing these fusion proteins were then used to transfect primary MECs. Transfected MECs were observed under fluorescence confocal microscopy. We found that OCLN frequently colocalized with BTN1a1 and XOR proteins, and they often migrated together inside the cells (Fig 6E and 6F, S1 Table, S2 and S3 Movies). Finally, we wanted to examine whether OCLN binding to BTN1a1 and XOR depended on the N-terminus or C-terminus cytoplasmic domain of OCLN [22,27]. To this end, we tagged the N- or C-terminus truncation of OCLN with FLAG peptides. Tagged OCLN truncations were then introduced into 293 cells via transfection of transient expression constructs, followed by co-IP to test their potential interactions with BTN1a1 and XOR, which were tagged with the HA peptides. We found that the removal of the C-terminus of OCLN almost completely abrogated its ability to bind either BTN1a1 or XOR, while the removal of the N-terminus of OCLN did not have such an effect. These data suggest that the C-terminus is an essential functional component of OCLN during the regulation of lipid secretion via binding to BTN1a1 and XOR. Taken together, the above data show that OCLN localizes to LDs and could directly participate in secretion by binding to BTN1a1 and XOR in MECs in a C-terminus–dependent manner.

[END]

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