golgi gets wired up

3
NATURE CELL BIOLOGY VOLUME 10 | NUMBER 8 | AUGUST 2008 885 NEWS AND VIEWS a major role in controlling endothelial barrier function. For example, VE-cadherin endocy- tosis and uncoupling from catenin family pro- teins contribute to the disruption of endothelial cell–cell junctions in response to VEGF 11,12 . In light of the study from Taddei et al. inhibition of VE-cadherin expression, its adhesive prop- erties or its plasma membrane availability may allow β-catenin and FoxO1 nuclear accumu- lation, and therefore repression of claudin-5 transcription (Fig. 1). Thus, although acute VEGF stimulation could induce an increase in vascular permeability by a reversible dis- ruption of VE-cadherin adhesion and sub- sequent disorganization of tight junctions 11 (Fig. 1b), chronic exposure to VEGF or other pro-permeability factors may severely affect the endothelial barrier, as the turnover and expres- sion of claudin-5 may be compromised in the absence of functional VE-cadherin adhesion (Fig. 1c). Regulation of claudin-5 expression by VE-cadherin points to crosstalk between tight and adherens junctions. This crosstalk is bi-directional, as tight junction molecules, such as JAMs, may regulate cadherin-mediated adhesion in endothelial cells 13 . Aside from VEGF, reactive oxygen species can increase vascular permeability through a Rac-dependent mechanism, associated with loss of endothelial cell–cell junctions 14 (Fig. 1c). Recent work in Caenorhabditis elegans sug- gests that oxygen-stress signalling depends on a functional interaction between FoxO and β-catenin 15 . From the findings of Taddei et al. we may speculate that reactive oxygen spe- cies could have an early effect on VE-cadherin adhesion through Rac signalling and a later effect through downregulation of claudin-5 expression (Fig. 1). This two-pronged control of endothelial junctions by VE-cadherin function may contribute to the pathological disruption of the vascular wall, which is observed in a variety of diseases, such as tumour-induced angiogen- esis, stroke, myocardial infarction, inflamma- tion, allergy and macular degeneration. It is also noteworthy that the severe blood- brain barrier phenotype of claudin-5 knockout mice has prompted the search for treatments of diseases associated with aberrant blood-brain barrier function, or those that enhance the effi- ciency of drug delivery to the brain 8 . On the basis of the signalling pathway characterized here, VE-cadherin could be considered as a molecular target for the treatment of central nervous system diseases. For example, the mor- bidity of stroke is associated with neural cell damage caused by the breakdown of the blood- brain barrier and the subsequent exudation of plasma components and oedema. In this regard, blocking VEGF pro-permeability signalling has successfully limited the size of lesions in mouse models of brain ischaemia 16 . We can now antic- ipate that restoring VE-cadherin adhesion and function may not only reduce vascular leakage, perhaps more effectively than VEGF antago- nists, but also help rescue blood-brain barrier function by providing positive feedback on claudin-5 expression. Vascular leakage and loss of endothelial barrier integrity is a hallmark of pathological angiogenesis. Substantial progress had been made recently in understanding the molecu- lar mechanisms regulating endothelial cell–cell junctions. The direct effect of VE-cadherin on the regulation of claudin-5 expression provides a further rationale for the development of ther- apeutic approaches targeting VE-cadherin and its downstream molecules for vascular ‘normal- ization’ in many human diseases that involve aberrant angiogenesis and vascular leakage. 1. Yap, A. S., Brieher, W. M. & Gumbiner, B. M. Annu. Rev. Cell .Dev. Biol. 13, 119–146 (1997). 2. Tsukita, S., Furuse, M. & Itoh, M. Nature Rev. Mol. Cell Biol. 2, 285–293 (2001). 3. Dejana, E. Nature Rev. Mol. Cell Biol. 5, 261–270 (2004). 4. Taddei, A. et al. Nature Cell Biol. 10, 923–934 (2008). 5. Nelson, W. J. & Nusse, R. Science 303, 1483–1487 (2004). 6. Rhee, J., Buchan, T., Zukerberg, L., Lilien, J. & Balsamo, J. Nature Cell Biol. 9, 883–892 (2007). 7. Hoogeboom, D. et al. J. Biol. Chem. 283, 9224–9230 (2008). 8. Nitta, T. et al. J. Cell Biol. 161, 653–660 (2003). 9. Carmeliet, P. et al. Cell 98, 147–57 (1999). 10. Lampugnani, M. G., Orsenigo, F., Gagliani, M. C., Tacchetti, C. & Dejana, E. J. Cell Biol. 174, 593–604 (2006). 11. Gavard, J. & Gutkind, J. S. Nature Cell Biol. 8, 1223– 1234 (2006). 12. Weis, S. et al. J. Clin. Invest. 113, 885–894 (2004). 13. Orlova, V. V., Economopoulou, M., Lupu, F., Santoso, S. & Chavakis, T. J. Exp. Med. 203, 2703–2714 (2006). 14. van Wetering, S. et al. J. Cell Sci. 115, 1837–1846 (2002). 15. Essers, M. A. G. et al. Science 308, 1181–1184 (2005). 16. Paul, R. et al. Nature Med. 7, 222–227 (2001). Golgi gets wired up Lennart Asp and Tommy Nilsson In endocrine cells, secretion can be rapidly upregulated in response to stimuli without the need for additional synthesis of transport components. A new and unexpected function of KDEL-R as a signalling receptor that senses cargo protein load in the early secretory pathway has been identified. Mammalian cells secrete biosynthetic cargo in response to both external and internal stimuli. It is known that in the early secretory pathway, anterograde (forward) transport of biosynthetic material from the endoplasmic reticulum (ER) to the Golgi is, at least to some degree, offset by retrograde transport through COPI vesicles and possibly, tubular transport intermediates 1 . Thus, the combined contribu- tion of anterograde and retrograde traffic will define the flux of cargo proteins through the secretory pathway. Retrograde carriers move components, including modifying enzymes, chaperones and transport machinery proteins, from downstream (late) Golgi compartments to upstream compartments, including the ER (see Fig. 1a). At the interface between the ER and the Golgi apparatus, components of the trafficking machinery exit the ER through Lennart Asp and Tommy Nilsson are in the Department of Medical Genetics, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden. e-mail: [email protected], [email protected] © 2008 Macmillan Publishers Limited. All rights reserved.

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nature cell biology volume 10 | number 8 | AuGuST 2008 885

N e w s a N d v i e w s

a major role in controlling endothelial barrier function. For example, VE-cadherin endocy-tosis and uncoupling from catenin family pro-teins contribute to the disruption of endothelial cell–cell junctions in response to VEGF11,12. In light of the study from Taddei et al. inhibition of VE-cadherin expression, its adhesive prop-erties or its plasma membrane availability may allow β-catenin and FoxO1 nuclear accumu-lation, and therefore repression of claudin-5 transcription (Fig. 1). Thus, although acute VEGF stimulation could induce an increase in vascular permeability by a reversible dis-ruption of VE-cadherin adhesion and sub-sequent disorganization of tight junctions11 (Fig. 1b), chronic exposure to VEGF or other pro-permeability factors may severely affect the endothelial barrier, as the turnover and expres-sion of claudin-5 may be compromised in the absence of functional VE-cadherin adhesion (Fig. 1c). Regulation of claudin-5 expression by VE-cadherin points to crosstalk between tight and adherens junctions. This crosstalk is bi-directional, as tight junction molecules, such as JAMs, may regulate cadherin-mediated adhesion in endothelial cells13.

Aside from VEGF, reactive oxygen species can increase vascular permeability through a Rac-dependent mechanism, associated with loss of endothelial cell–cell junctions14 (Fig. 1c). Recent work in Caenorhabditis elegans sug-gests that oxygen-stress signalling depends on a functional interaction between FoxO and β-catenin15. From the findings of Taddei et al.

we may speculate that reactive oxygen spe-cies could have an early effect on VE-cadherin adhesion through Rac signalling and a later effect through downregulation of claudin-5 expression (Fig. 1). This two-pronged control of endothelial junctions by VE-cadherin function may contribute to the pathological disruption of the vascular wall, which is observed in a variety of diseases, such as tumour-induced angiogen-esis, stroke, myocardial infarction, inflamma-tion, allergy and macular degeneration.

It is also noteworthy that the severe blood-brain barrier phenotype of claudin-5 knockout mice has prompted the search for treatments of diseases associated with aberrant blood-brain barrier function, or those that enhance the effi-ciency of drug delivery to the brain8. On the basis of the signalling pathway characterized here, VE-cadherin could be considered as a molecular target for the treatment of central nervous system diseases. For example, the mor-bidity of stroke is associated with neural cell damage caused by the breakdown of the blood-brain barrier and the subsequent exudation of plasma components and oedema. In this regard, blocking VEGF pro-permeability signalling has successfully limited the size of lesions in mouse models of brain ischaemia16. We can now antic-ipate that restoring VE-cadherin adhesion and function may not only reduce vascular leakage, perhaps more effectively than VEGF antago-nists, but also help rescue blood-brain barrier function by providing positive feedback on claudin-5 expression.

Vascular leakage and loss of endothelial barrier integrity is a hallmark of pathological angiogenesis. Substantial progress had been made recently in understanding the molecu-lar mechanisms regulating endothelial cell–cell junctions. The direct effect of VE-cadherin on the regulation of claudin-5 expression provides a further rationale for the development of ther-apeutic approaches targeting VE-cadherin and its downstream molecules for vascular ‘normal-ization’ in many human diseases that involve aberrant angiogenesis and vascular leakage.

1. Yap, A. S., Brieher, W. M. & Gumbiner, B. M. Annu. Rev. Cell .Dev. Biol. 13, 119–146 (1997).

2. Tsukita, S., Furuse, M. & Itoh, M. Nature Rev. Mol. Cell Biol. 2, 285–293 (2001).

3. Dejana, E. Nature Rev. Mol. Cell Biol. 5, 261–270 (2004).

4. Taddei, A. et al. Nature Cell Biol. 10, 923–934 (2008).

5. Nelson, W. J. & Nusse, R. Science 303, 1483–1487 (2004).

6. Rhee, J., Buchan, T., Zukerberg, L., Lilien, J. & Balsamo, J. Nature Cell Biol. 9, 883–892 (2007).

7. Hoogeboom, D. et al. J. Biol. Chem. 283, 9224–9230 (2008).

8. Nitta, T. et al. J. Cell Biol. 161, 653–660 (2003).9. Carmeliet, P. et al. Cell 98, 147–57 (1999).10. Lampugnani, M. G., Orsenigo, F., Gagliani, M. C.,

Tacchetti, C. & Dejana, E. J. Cell Biol. 174, 593–604 (2006).

11. Gavard, J. & Gutkind, J. S. Nature Cell Biol. 8, 1223–1234 (2006).

12. Weis, S. et al. J. Clin. Invest. 113, 885–894 (2004).13. Orlova, V. V., Economopoulou, M., Lupu, F., Santoso,

S. & Chavakis, T. J. Exp. Med. 203, 2703–2714 (2006).

14. van Wetering, S. et al. J. Cell Sci. 115, 1837–1846 (2002).

15. Essers, M. A. G. et al. Science 308, 1181–1184 (2005).

16. Paul, R. et al. Nature Med. 7, 222–227 (2001).

Golgi gets wired upLennart Asp and Tommy Nilsson

in endocrine cells, secretion can be rapidly upregulated in response to stimuli without the need for additional synthesis of transport components. A new and unexpected function of KdeL-R as a signalling receptor that senses cargo protein load in the early secretory pathway has been identified.

Mammalian cells secrete biosynthetic cargo in response to both external and internal stimuli. It is known that in the early secretory

pathway, anterograde (forward) transport of biosynthetic material from the endoplasmic reticulum (ER) to the Golgi is, at least to some degree, offset by retrograde transport through COPI vesicles and possibly, tubular transport intermediates1. Thus, the combined contribu-tion of anterograde and retrograde traffic will define the flux of cargo proteins through the

secretory pathway. Retrograde carriers move components, including modifying enzymes, chaperones and transport machinery proteins, from downstream (late) Golgi compartments to upstream compartments, including the ER (see Fig. 1a). At the interface between the ER and the Golgi apparatus, components of the trafficking machinery exit the ER through

Lennart Asp and Tommy Nilsson are in the Department of Medical Genetics, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden. e-mail: [email protected], [email protected]

© 2008 Macmillan Publishers Limited. All rights reserved.

886 nature cell biology volume 10 | number 8 | AuGuST 2008

N e w s a N d v i e w s

COPII-coated vesicles, together with biosyn-thetic cargo, and therefore need to be recycled to the ER to enable subsequent rounds of trans-port. Mechanistically, this is achieved through retrieval signals, which, for membrane proteins, are composed of basic cytoplasmic amino acids that make up the canonical sequence K(X)KXX-COOH. This sequence binds to COPI-coat proteins, directly ensuring recycling to the ER. Similarly, many luminal ER proteins are equipped with a C-terminal signal, KDEL-COOH, which binds to a receptor termed the KDEL receptor (KDR) located in the cis-part of the Golgi apparatus. On binding of a KDEL-containing protein, the KDR oligomerizes, enters budding COPI vesicles and is recycled

back to the ER. It has long been alleged that retrograde transport is self-balanced such that if proteins need to be recycled, retrograde car-riers form as a consequence. This direct link is easy to envisage for membrane proteins that contain a -K(X)KXX-COOH signal but not for the KDR, which lacks such a recycling signal. Instead, evidence suggests that the KDR can be phosphorylated on Ser 209 and that this results in the binding of COPI-coat proteins2. On binding of KDEL proteins, the KDR recep-tor also oligomerizes3, raising the possibility that a link between ligand-mediated KDR oligomerization and phosphorylation ensures recruitment of COPI-coat proteins, and entry into recycling COPI vesicles.

In this issue, Pulvirenti et al.4 show that an increased protein load entering the cis-Golgi from the ER activates Src kinases through the KDR and that this signalling regulates overall transport activity in the secretory pathway. As such, Src kinases can now be added to the list of important signalling molecules, which includes cAMP5, phosphatidylinositol 4-phosphate6, diacylglycerol7, protein kinase D8, ras9, cdc42 (ref. 10), trimeric G proteins11 and IRE1/ATF6/PERK12, that operate in the secretory pathway. The last of these regulate the unfolded protein response (UPR), a mechanism used by the cell to respond rapidly to an increased level of unfolded proteins in the ER. This involves luminal chaperones that bind transmembrane receptors termed IRE1, ATF6 or PERK when levels of unfolded polypeptides are relatively low. Under conditions of stress, such as heat shock or viral replication, there is an immedi-ate imbalance between the amount of unfolded proteins and available chaperones. This is sensed by the IRE1/ATF6/PERK signalling net-work and, through pre-mRNA processing of a nuclear transcription factor, leads to increased expression of ER chaperones, lipid synthesis enzymes, as well as endoplasmic reticulum-associated degradation (ERAD) proteins. In addition, IRE1/ATF6/PERK can downregu-late protein synthesis by removing ER-bound mRNAs and trigger apoptosis. All three events are part of the UPR and have as a central theme: the ability to sense the level of (unfolded) cargo load in the ER lumen12.

Pulvirenti et al. show that the ability to sense increased cargo load at the Golgi apparatus can be attributed to the KDEL-KDR-Src axis. Their study shows that silencing of KDR or Src by siRNA, expression of KDR mutants or addi-tion of Src kinase inhibitors, result in an over-all downregulation of anterograde transport. This suggests that Src kinases are required to phosphorylate components of the trafficking machinery in response to elevated levels of pro-tein transport passing through the cis-Golgi cis-ternae. Alternatively, or additionally, inhibition of KDR or Src function may also result in the reduced ability to recycle components needed for ER-to-Golgi transport13. This would be through a direct downregulation of recycling COPI vesicles. Through two-hybrid interac-tion studies, Pulvirenti et al. provide evidence that Src is directly bound to the KDR. Also, the observation that expression of constitutive active mutants of Src kinase reduces the inhibi-tory effect of dominant-negative KDR mutants

Anterograde

Retrograde

COP I

COP II

VTC cisGolgi

medialGolgi

transGolgi

ER

ER

KDR-binding protein

Foldedprotein

UPR

Src

Src

Src Src

KDELreceptor

Unfoldedprotein

Figure 1 Schematic overview of the secretory pathway. Anterograde transport is initiated at the level of the ER where newly synthesized proteins exit the ER through budding COPII vesicles. Unfolded proteins usually mature in the ER and leave as folded proteins, having passed an ER-based quality control system. Proteins maturation then occurs in the Golgi apparatus. For example, O-linked glycosylation, which is initiated in the Golgi apparatus, markedly affects the maturation and function of proteins. Similarly, processing of N-linked oligosaccharides in the Golgi apparatus can dictate final function of proteins exiting the secretory pathway. Some proteins also enter the cis-Golgi only to be returned to the ER for final folding/maturation. The formyl glycine-generating enzyme, a catalytic enzyme essential for the activation of sulphotransferases, interacts with the RDEL-containing and cis-Golgi resident Erp44, which returns the enzyme through the KDR to the ER for its final folding/maturation16. Other luminal KDR-binding proteins may also escape the ER on increased trafficking and together with Golgi resident proteins, such as Erp44, trigger a Src kinase signalling cascade when binding to the KDR. This then results in the formation of retrograde COPI vesicles and transport to the ER. Src-based signalling may also lead to upregulation of the activity of other transport components that together, enables the secretory pathway to cope with an increased cargo-load while at the same time, maintaining high fidelity in protein folding and maturation. The depicted Src-mediated signalling through the KDR is mirrored by the UPR that signals the cell to produce more ER chaperones in response to a marked increase in the amount of unfolded proteins in the ER. Note that the Golgi apparatus can also exchange material with the ER through its trans side. This is made possible by the close proximity of ER membranes that interdigitates the trans-most cisternae of the Golgi stack. This allows for a coat-independent exchange of material between the Golgi and the ER of glycosylation enzymes, enterotoxins and possibly, lipids (VTC, vesicular tubular clusters).

© 2008 Macmillan Publishers Limited. All rights reserved.

nature cell biology volume 10 | number 8 | AuGuST 2008 887

N e w s a N d v i e w s

further indicates a direct functional relationship between Src and KDR. As KDRs oligomerize on binding of KDEL-containing proteins, a sim-ple mechanism of Src kinase activation through ligand-mediated KDR oligomerization is there-fore possible (Fig. 1b). Together with an unregu-lated tyrosine phosphate, this would constitute a robust core signalling system sufficient to mediate Src kinase signal transduction through ligand-mediated receptor oligomerization14.

A Src kinase bound to the KDR can either phosphorylate the cytoplasmic domain of the KDR or phosphorylate Src kinases bound to other KDRs. In the case of the former, binding of COPI-coat proteins would then be a direct consequence; in the case of the latter, activated Src kinases would now phosphorylate multiple targets. Indeed, there is already evidence for a feed-forward gating of COPI-vesicle forma-tion through Src-mediated tyrosine phospho-rylation of atypical protein kinase C ι/λ and glycerylaldehyde-3-phosphate dehydrogenase (GAPDH)15. When phosphorylated, atypical

protein kinase C ι/λ and GAPDH interact and form a tripartite complex with rab2, which binds to vesicular tubular clusters (VTC) and cis-Golgi membranes to stimulate COPI-vesicle formation. Thus, an increase in proteins bear-ing the KDEL-COOH signal associated with unfolded biosynthetic cargo may stimulate a corresponding increase in retrograde traf-ficking by COPI vesicles through such a Src-mediated signalling cascade. It is likely that this constitutes only one of many possible models and it will now be interesting to identify which other components of the secretory pathway are phosphorylated directly or indirectly by Src kinases, and what consequence this has for their function. Does this involve COPI-vesicle formation from Golgi cisternae other than the cis-Golgi or does Src phosphoryla-tion promote the formation of tubular trans-port intermediates and tubular inter-cisternal connections, both of which are associated with high transport activity? These and additional avenues regulated by Src and other signalling

molecules now need to be explored before we can fully understand the workings of the secre-tory pathway.

1. Rabouille, C. & Klumperman, J. Nature Rev. Mol. Cell Biol. 6, (10), 812–817 (2005).

2. Cabrera, M. et al. Mol. Biol. Cell 14, (10), 4114–4125 (2003).

3. Majoul, I. et al. Dev. Cell 1, 139–153 (2001).4. Pulvirenti, T. et al. Nature Cell Biol. 10, 912–922

(2008).5. Muniz, M. et al. J. Biol. Chem. 271, 30935–30941

(1996).6. D’Angelo, G. et al. J. Cell Sci. 121, 1955–1963

(2008).7. Litvak, V. et al. Nature Cell Biol. 7, 225–234 (2005).8. Baron, C. L. & Malhotra, V. Science 295, 325–

328(2002). 9. Bivona, T. G. et al. Nature 424, 694–698 (2003).10. Chen, J. L. et al. J. Cell Biol. 169, 383–389 (2005).11. Donaldson, J. G. et al. Science 254, 1197–1199

(1991). 12. Ron, D. & Walter, P. Nature Rev. Mol. Cell Biol. 8, 519–

529 (2007).13. Bard, F. et al. J. Biol. Chem. 278, 46601–46606

(2003).14. Cooper J. A. & Qian, H. Biochemistry 47, 5681– 5688

(2008).15. Tisdale, E. J. & Artalejo, C. R. Traffic 8, 733–741

(2007).16. Mariappan, M. et al. J. Biol. Chem. 283, 6375– 6383

(2008).

Cytokine loops driving senescenceJiri Bartek, Zdenek Hodny and Jiri Lukas

Cellular senescence, the permanent state of cell-cycle arrest, is emerging as an intrinsic barrier against tumorigenesis and a mechanism contributing to organismal ageing. Unexpected findings now identify multiple secreted inflammatory cytokines, their cognate receptors and positive-feedback loops with corresponding transcription factors, as key mediators of both oncogene-induced and replicative senescence.

Cellular senescence, a state of irreversible withdrawal of cells from the proliferative pool, occurs on exhaustion of the proliferative lifespan (so-called replicative senescence), in response to activated oncogenes (oncogene-induced senescence) or on exposure of cells to DNA-damaging insults or other stresses1,2 (premature senescence). The biological signif-icance of cellular senescence is probably best documented by its in vivo role as an intrinsic mechanism preventing tumour progression from oncogene-transformed, premalignant

cells1,2,3. At the same time, this critical tumour-suppressive mechanism comes at a price for the organism, as it limits the regenerative capacity of normal tissue stem cells and hence eventu-ally contributes to organismal ageing1,2. Given these fundamental physiological roles, it is not surprising that elucidation of the molecular basis of cellular senescence has been among the priorities of contemporary biomedical research. Two studies published in Cell4,5 now extend recent findings in this field, by report-ing that secreted inflammatory cytokines are crucial mediators of senescence.

In one of these two studies, Acosta et al.4 used an unbiased loss-of-function screen to identify genes required for replicative senes-cence of primary human fibroblasts. The authors found that knockdown of the chem-

okine receptor CXCR2 (also known as IL8RB) prolonged the lifespan of near-senescent IMR-90 cells, and further validation confirmed that CXCR2 was necessary for both replicative and oncogene-induced senescence. Conversely, ectopic expression of CXCR2 induced prema-ture senescence in a p53-dependent manner. Acosta et al. also showed that cells undergoing H-Ras and MEK oncogene-induced senescence secreted numerous inflammatory chemokines known to interact with CXCR2, including GROα and the interleukins IL-1, IL-6 and IL-8. The increased levels of these ligands, as well as that of the CXCR2 receptor during senescence were attributed to transcriptional upregulation by the NF-κB and C/EBPβ transcription fac-tors, which were themselves upregulated in senescent cells. Together, these results suggest

Jiri Bartek, Zdenek Hodny and Jiri Lukas are in the Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Copenhagen, Denmark, and in the Laboratory of Genome Integrity, Institute of Molecular Genetics, the Czech Academy of Sciences, Prague, Czech Republic. e-mail: [email protected]; [email protected]; [email protected]

© 2008 Macmillan Publishers Limited. All rights reserved.