Wendy R. Kam

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro- translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endo-somal sorting of the toxin-GM1 complex.

Transcription, Translation, and Function of Lubricin, a Boundary Lubricant, at the Ocular Surface

IMPORTANCE Lubricin may be an important barrier to the development of corneal and conjunctival epitheliopathies that may occur in dry eye disease and contact lens wear. OBJECTIVE To test the hypotheses that lubricin (ie, proteoglycan 4 [PRG4 ]), a boundary lubricant, is produced by ocular surface epithelia and acts to protect the cornea and conjunctiva against significant shear forces generated during an eyelid blink and that lubricin deficiency increases shear stress on the ocular surface and promotes corneal damage. DESIGN, SETTING, AND PARTICIPANTS Human, porcine, and mouse tissues and cells were processed for molecular biological, immunohistochemical, and tribological studies, and wild-type and PRG4 knockout mice were evaluated for corneal damage. RESULTS Our findings demonstrate that lubricin is transcribed and translated by corneal and conjunctival epithelial cells. Lubricin messenger RNA is also present in lacrimal and meibomian glands, as well as in a number of other tissues. Absence of lubricin in PRG4 knockout mice is associated with a significant increase in corneal fluorescein staining. Our studies also show that lubricin functions as an effective friction-lowering boundary lubricant at the human cornea-eyelid interface. This effect is specific and cannot be duplicated by the use of hyaluronate or bovine serum albumin solutions. CONCLUSIONS AND RELEVANCE Our results show that lubricin is transcribed, translated, and expressed by ocular surface epithelia. Moreover, our findings demonstrate that lubricin presence significantly reduces friction between the cornea and conjunctiva and that lubricin deficiency may play a role in promoting corneal damage.

A Single Native Ganglioside GM1-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

ABSTRACT Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM1 (GM1) molecules, and the toxin-GM1 complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM1 by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476–1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573–586, 2012), suggesting that multivalent binding to GM1 is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM1 binding sites from zero (nonbinding) to five (wild type). We found that a single GM1 binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM1 receptors is not essential for toxicity of CT. IMPORTANCE Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB5 subunit toxins that can bind multivalently to cell membrane lipid receptors. Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB5 subunit toxins that can bind multivalently to cell membrane lipid receptors.

Effect of Azithromycin on Lipid Accumulation in Immortalized Human Meibomian Gland Epithelial Cells

Meibomian gland dysfunction (MGD) is believed to be the leading cause of dry eye disease (DED), which afflicts tens of millions Americans (1). Of particular interest, the most common pharmaceutical treatment for the management of MGD in the United Statesis the off-label use of topical azithromycin (2). This macrolide antibiotic is presumed to be effective because of its anti-inflammatory and anti-bacterial actions, which may suppress the MGD-associated posterior blepharitis and growth of lid bacteria (3). However, there are no published, peer-reviewed data demonstrating that azithromycin has the ability to act directly on the human meibomian gland to enhance this tissue’s function, and to ameliorate the pathophysiology of MGD. We hypothesize that azithromycin can act directly on human meibomian gland epithelial cells to stimulate their differentiation, enhance the quality and quantity of their lipid production, and promote their holocrine secretion. Our purpose was to begin to test our hypothesis.

Effect of Growth Factors on the Proliferation and Gene Expression of Human Meibomian Gland Epithelial Cells

PURPOSE We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses. METHODS Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures. RESULTS Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. CONCLUSIONS Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis.

The Influence of 13-cis Retinoic Acid on Human Meibomian Gland Epithelial Cells

PURPOSE Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells. METHODS To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated. RESULTS We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9. CONCLUSIONS Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA-related induction of MGD.

One man's poison is another man's meat: Using azithromycin-induced phospholipidosis to promote ocular surface health

Drug-induced phospholipidosis (PLD) is a common adverse effect which has led to the termination of clinical trials for many candidate pharmaceuticals. However, this lipid-inducing effect may be beneficial in the treatment of meibomian gland dysfunction (MGD). MGD is the major cause of dry eye disease (DED), which affects 40 million people in the USA and has no cure. Azithromycin (AZM) is a PLD-inducing antibiotic that is used off-label to treat MGD, and is presumably effective because it suppresses the MGD-associated conjunctival inflammation (i.e. posterior blepharitis) and growth of lid bacteria. We hypothesize that AZM can act directly to promote the function of human meibomian gland epithelial cells by inducing PLD in these cells, characterized by the accumulation of lipids and lysosomes. Immortalized human meibomian gland epithelial cells (HMGEC) were cultured with or without azithromycin for 5 days. Cells were evaluated for cholesterol (Filipin) and neutral lipid (LipidTox) staining, as well as the appearance of lysosomes (LysoTracker) and lamellar bodies (transmission electron microscopy, TEM). The lipid composition of cellular lysates was analyzed by high performance thin-layer chromatography. Our findings demonstrate that AZM stimulates the accumulation of free cholesterol, neutral lipids and lysosomes in HMGEC. This AZM-induced increase of neutral lipid content occurred predominantly within lysosomes. Many of these vesicles appeared to be lamellar bodies by TEM, which is the characteristic of PLD. Our findings also show that AZM promotes an accumulation of free and esterified cholesterol, as well as phospholipids in HMGECimmortalized. Our results support our hypothesis and confirm the beneficial effect of PLD induced by AZM on HMGEC. Our discovery reveals a new potential use of PLD-inducing drugs, and makes this adverse effect a beneficial effect.

Neurotransmitter Influence on Human Meibomian Gland Epithelial Cells

PURPOSE A striking characteristic of the human meibomian gland is its rich sensory, sympathetic, and parasympathetic innervation, yet the functional relevance of these nerve fibers remains unknown. Acting on the hypothesis that neurotransmitters are released in the vicinity of the gland, act on glandular receptors, and influence the production, secretion, and/or delivery of meibomian gland secretions to the ocular surface, the goal in this study was to begin to determine whether neurotransmitters influence the meibomian gland. METHODS Immortalized human meibomian gland epithelial (SLHMG) cells were examined for the presence of vasoactive intestinal peptide (VIP) and muscarinic acetylcholine (mACh) receptor transcripts and proteins. Cells were also exposed to VIP, carbachol, forskolin, and/or 3-isobutyl-1-methylxanthine (IBMX) to determine whether these agents, alone or in combination, modulate the adenylyl cyclase pathway, the accumulation of intracellular free calcium ([Ca2+]i), or cell proliferation. RESULTS Results demonstrate that SLHMG cells transcribe and translate VIP and mACh receptors; VIP, with either IBMX or forskolin, activates the adenylyl cyclase pathway, and the effect of VIP and forskolin together is synergistic; both VIP and carbachol increase intracellular [Ca2+] in SLHMG cells; and VIP with forskolin stimulates SLHMG cell proliferation. CONCLUSIONS This study shows that parasympathetic neurotransmitters and their agonists influence the function of human meibomian gland epithelial cells. It remains to be determined whether this action alters the production, secretion, and/or delivery of meibum to the ocular surface.

N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.

Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

PURPOSE We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. METHODS Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. RESULTS Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. CONCLUSIONS Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland.