| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Division of Neuroscience and Mental Health, Imperial College, London, UK;
Department Human Anatomy and Genetics, University of Oxford, Oxford, UK; and
Department of Biochemical Pharmacology, William Harvey Research Institute, London, UK
1 Correspondence: Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Imperial College London, Hammersmith Campus, Du Cane Rd., London W12 0NN, UK. E-mail: e.solito{at}imperial.ac.uk
SPECIFIC AIMS
Annexin 1(ANXA1) has an important role in cell-cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane-bound receptors of adjacent cells after translocation of the protein from the cytoplasm to the cell surface. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo-stellate) cells induced by LPS, focusing on the roles of: 1) serine phosphorylation and 2) lipidation.
PRINCIPAL FINDINGS
1. Characterization of the TtT/GF cells
LPS is the major mediator of the organism’s response to Gram-negative bacterial invasion. In the pituitary gland, LPS targets the folliculo-stellate (FS) causing them to release interleukin (IL)-6 and other cytokines that act locally to modulate the secretory activity of the endocrine cells and thereby play an important part in mediating the neuroendocrine response to bacterial infection. We have previously shown that the inhibitory effects of glucocorticoids on ACTH release from the corticotrophs are partially dependent on the externalization of ANXA1 from FS cells. Other data indicate that IL-6 up-regulates the transcriptional activity of the Anxa1 promoter, raising the possibility that LPS may also influence the cellular disposition of ANXA1 in FS cells. To explore this possibility, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and FACS analysis to confirm that TtT/GF cells express the necessary machinery to respond to LPS and demonstrated expression mRNA and protein for CD14, TLR2, and TLR4.
2. Role of phosphorylation in the membrane translocation of ANXA1
LPS (100 ng/ml) increased the expression of Anxa1 mRNA and ANXA1 protein in the cytoplasm, as determined by RT-PCR and Western blot analysis; its effects were first evident after 8 h contact and declined thereafter. By contrast, LPS had no effect on the cell surface expression of ANXA1 protein. Together these data suggest that LPS regulates the synthesis but not the cellular disposition of ANXA1. The antibody (Ab) employed for these studies did not distinguish phosphorylated and unphosphorylated species of ANXA1. Subsequently, we used an Ab, which recognizes ANXA1 only when phosphorylated on serine 27 (ANXA1-S27-PO4) and revealed a different profile of data. LPS induced a marked increase in ANXA1-S27-PO4 on the cell surface within 5 min of contact. Time-course and pharmacological studies revealed that the response was transient (<30 min) and dependent on PI3-kinase and MAP-kinase, but not IL-6. Treatment of the cell surface protein fraction with alkaline phosphatase reduced its ANXA1-S27-PO4 content, confirming the specificity of the Ab.
To substantiate our finding that serine phosphorylation is essential for the membrane translocation of ANXA1, we developed an enhanced GFP (EGFP)-tagged ANXA1 construct in which serine was replaced with alanine. Microscopic analysis showed strong expression of the control [wild-type (WT)] EGFP-ANXA1 construct in the cytoplasm and nucleus of TtT/GF cells. The tagged protein translocated to the plasma membrane, accumulating at one pole, and also to the nuclear membrane in stimulation with LPS (Fig. 1
A and B, row 2). The mutant construct (EGFP-ANXA1-S27/A) also showed strong cytoplasmic and nuclear expression (Fig. 1A
, row 3). However, unlike EGFP-ANXA1, it accumulated in patches at a submembrane location as well as in the nucleus on exposure to LPS (Fig. 1B
, row 3). The submembrane accumulation was confirmed by 1) costaining with phallodin and 2) deconvolution xz reconstruction (0.5 µm), which demonstrated the contrasting membrane localization of ANXA-wild-type-enhanced GFP and punctuate submembrane distribution of ANXA1-S27/A-enhanced GFP after LPS stimulation.
|
3. Role of lipidation in the translocation of ANXA1 to the cell membrane
Lipidation is involved in the maturation of many proteins in prokaryotic and eukaryotic cells. Both myristoylated and prenylated proteins interact electrostatically with the negative phospholipid head groups in the lipid bilayer to fine-tune the association with the membrane. ANXA1 is well known to interact with negatively charged membrane phospholipids and inspection of its sequence included potential sites for both myristoylation and prenylation. To explore the potential role of lipidation in the translocation of ANXA1 to the cell surface, we examined the effects of lovastatin (an inhibitor of HMG-coenzyme A, 10 mM) on the response to LPS (100 ng/ml, 5 min). Lovastatin abolished the significant increase of cell surface ANXA1-S27-PO4 induced by LPS, as determined by FACS. Its effects were overcome by mevalonic acid (the product of HMG-CoA, 10 mM) and farnesylpyrophosphate (a substrate for protein farnesylation, FPP 5 µM) but not by geranyl-geranylpyrophosphate (GGPP, 5 µM) or cholesterol (200 µM). These data were confirmed by Western blot analysis. In addition, the LPS-induced translocation of ANXA1-S27-PO4 to the cell surface was prevented by treatment of the cells with 2-hydroxymyristic acid (an inhibitor of myristoylation, 1 mM, 16 h).
CONCLUSIONS AND SIGNFICANCE
LPS is a potent activator of the HPA axis, exerting its effects at multiple loci within the axis, including the pituitary FS cells. These cells express the LPS receptor TLR-4 and respond to the toxin by releasing cytokines (e.g., IL-6), which augments the ACTH response to the CRH. In the same vein, the FS cell line used here (TtT/GF) expresses the signaling molecules necessary to respond to LPS. ANXA1 is an important paracrine/juxtacrine mediator of the negative feedback actions of glucocorticoids in the hypothalamo-pituitary-adrenocortical (HPA) axis, particularly in conditions of immune/inflammatory insults; its actions in the pituitary gland are dependent on translocation of the protein from the cytoplasm to the cell surface of FS cells. Our finding that LPS mimics the actions of glucocorticoids in promoting ANXA1 release from our FS cell line is thus paradoxical. However, the temporal characteristics of the responses are different, with the toxin triggering a rapid, transient increase in membrane ANXA1-S27-PO4 and the steroids inducing a response, which is relatively slow in onset but sustained for several hours. As any increase in membrane ANXA1-S27-PO4 would be expected to suppress ACTH release, the transient response to LPS may contribute to the delay in the HPA response to the toxin that is evident in vivo. It may thus form part of the protective mechanism, which allows the proinflammatory host defense response to develop before it is contained within appropriate limits by the hypersecretion of the antiinflammatory glucocorticoids.
Our data obtained by FACS and Western blot analysis using an Ab, which specifically recognizes ANXA1-S27-PO4 showed clearly that serine phosphorylation is essential for the membrane translocation and externalization of ANXA1. They thus accord with studies in which ANXA1 release was elicited by glucocorticoids. The requirement of serine phosphorylation for the cellular exportation of ANXA1 was confirmed by immunofluorescence analysis of the distribution of wild type and mutant GFP-tagged ANXA1 proteins. The finding that the WT GFP-fused protein was distributed throughout the cytoplasm and that it translocated to the plasma membrane on stimulation with LPS, with preferential accumulation at one pole of cells is important as it demonstrates that the trafficking of GFP-tagged ANXA1 mirrors that of the native protein and, thus, that the tagged proteins are valid markers of the native protein. Importantly, our data also revealed that the mutant ANXA1-S27/A-EGFP protein shows a very different distribution in the cells, particularly after LPS stimulation when the mutant protein accumulates beneath the plasma membrane in a "patch-like" formation. These findings, together with the data from Western blot and FACS analysis, provide firm evidence that serine phosphorylation is critical to the translocation of ANXA1 to the cell surface. They also support the premise that both PI3-kinase and MAP-kinase are critical to this event as also is PKC. Further studies are now required to ascertain whether phosphorylation at other sites (e.g., serine) is also required.
A further important novel finding was that blockade of HMG-CoA reductase with lovastatin prevents the LPS-induced membrane translocation of ANXA1-S27-PO4. This effect was specific (it was overcome by mevalonic acid, the product of HMG-CoA reductase) and thus points for the first time to a role for lipidation in the translocation of ANXA1 to the cell surface. Since the effects of lovastatin were overcome by both FPP and 2-hydroxymyristic acid (although not by GGPP and cholesterol), both prenylation and myristoylation may important in this regard. The target(s) for lipidation remain to be identified. The sequence of ANXA1 itself includes potential sites for both modifications. However, the addition of lipid moieties would be expected to alter the molecular mass and electrophoretic mobility ANXA1 and no such changes were evident in our Western blot analyses. Thus, although we cannot exclude the possibility that the lipidation of ANXA1 is a transient event, perhaps essential for the passage of ANXA1 across the plasma membrane, it seems more likely that the targets are signaling intermediates, for example RAS and PKC, which are substrates for farnesylation and myristoylation respectively.
In conclusion, this study provides molecular, microscopic, and pharmacological evidence to support the premise that the trafficking of ANXA1 from the cytoplasm to the cell surface is dependent on post-translational modification and, specifically, phosphorylation of serine. It also provides novel evidence to suggest that lipidation is an important component of the signaling sequence effecting the membrane translocation of ANXA1-S27-PO4.
|
FOOTNOTES
2 Current address: Department Pharmaceutical Sciences, Faculty of Pharmacy, University of Salerno, Italy. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5319fje
This article has been cited by other articles:
|
|
 |
|
  N.-J. Hu, J. Bradshaw, H. Lauter, J. Buckingham, E. Solito, and A. Hofmann Membrane-Induced Folding and Structure of Membrane-Bound Annexin A1 N-Terminal Peptides: Implications for Annexin-Induced Membrane Aggregation Biophys. J., March 1, 2008; 94(5): 1773 - 1781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Vong, F. D'Acquisto, M. Pederzoli-Ribeil, L. Lavagno, R. J. Flower, V. Witko-Sarsat, and M. Perretti Annexin 1 Cleavage in Activated Neutrophils: A PIVOTAL ROLE FOR PROTEINASE 3 J. Biol. Chem., October 12, 2007; 282(41): 29998 - 30004. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Genge, L. N. Y. Wu, and R. E. Wuthier In Vitro Modeling of Matrix Vesicle Nucleation: SYNERGISTIC STIMULATION OF MINERAL FORMATION BY ANNEXIN A5 AND PHOSPHATIDYLSERINE J. Biol. Chem., September 7, 2007; 282(36): 26035 - 26045. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
