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to pore sizes of more than 9 nm were occasionally observed, demonstrating that the peroxisomal translocon could widen enough to accommodate the 9-nm PTS1-decorated gold particles previ-ously observed to be imported into peroxisomes3. Subsequent experiments analysing the connec-tivity of gating events showed that the peroxiso-mal import pore consists of one highly dynamic channel, in distinct contrast to the protein import pores of other organelles that have been reported to encompass multiple coupled pores within one complex. The data suggest that the peroxisomal import pore shifts between two different states: an inactive, stand-alone membrane complex and an active, dynamic and highly conductive transloca-tion pore when this membrane complex associ-ates with Pex5p linked to its cargo (Fig. 1).

This discovery, as is always the case, raises several questions: Which proteins form the flexible portion of the channel and how do they flex? What is the channel response to PTS2 cargo and the PTS2 receptor, Pex7p? How many receptor–cargo complexes can the channel accommodate at the same time? Can an in vitro system be established that allows for the true import of a peroxisomal matrix pro-tein into the lumen of an artificial lipid vesi-cle? Whatever the answers to these questions will be, there is no denying that the distinc-tive nature of the peroxisomal translocon is in keeping with the nonconformist reputation of that bad-boy organelle, the peroxisome.

CoMpeting FinAnCiAl inteRestsThe authors declare no competing financial interests.

1. Tabak, H. F., van der Zand, A. & Braakman, I. Curr. Opin. Cell Biol. 20, 393–400 (2008).

2. Glover, J. R., Andrews, D. W. & Rachubinski, R. A. Proc. Natl Acad. Sci. USA 91, 10541–10545 (1994).

3. Walton, P. A., Hill, P. E. & Subramani, S. Mol. Biol. Cell 6, 675–683 (1995).

4. Meinecke, M. et al. Nature Cell Biol. 12, 273–277 (2010).

5. McNew, J. A. & Goodman, J. M. Trends Biochem. Sci. 21, 54–58 (1996).

6. Schnell, D. J. & Hebert, D. N. Cell 112, 491–505 (2003).

7. Erdmann, R. & Schliebs, W. Nature Rev. Mol. Cell Biol. 6, 738–742 (2005).

8. Labarca, P., Wolff, D., Soto, U., Necochea, C. & Leighton, F. J. Membr. Biol. 94, 285–291 (1986).

9. Girzalsky, W., Saffian, D. & Erdmann, R. Biochim. Biophys. Acta doi.org/10.1016/j.bbamcr.2010.01.002).

10. Van der Laan, M. et al. Nature Cell Biol. 9, 1152–1159 (2007).

11. Koehler, C. M. Annu. Rev. Cell Dev. Biol. 20, 309–335 (2004).

12. Soll, J. & Schlieff, E. Nature Rev. Mol. Cell Biol. 5, 198–208 (2004).

PTH battles TGF‑β in boneAzeddine Atfi and Roland Baron

Bone remodelling in vertebrates is coordinately regulated by the opposing effects of parathyroid hormone (PTH) and transforming growth factor‑beta (TGF‑β). PTH couples the processes of bone resorption and formation by enforcing simultaneous internalization of TGF‑β type ii receptor (TβRii) and PTH type 1 receptor (PTH1R), which attenuates both TGF‑β and PTH signalling in vivo.

Skeletal development and homeostasis are regulated by a signalling network that bal-ances bone formation by osteoblasts with bone resorption by osteoclasts1. Perturbations of this network are associated with skeletal disorders such as osteoporosis, and an inher-ent risk of fracture and associated morbidity and mortality1. Examination of the molecu-lar mechanisms that govern bone formation during development and bone remodelling in adults has revealed a role for the PTH and TGF-β families of signalling molecules. PTH acts both as a trophic and anabolic hormone that fosters osteoblast proliferation, survival and differentiation2,3, whereas TGF-β favours

osteoblast proliferation but restricts osteoblast maturation, mainly by repressing the expres-sion of genes involved in bone formation4,5.

Although PTH and TGF-β are well known to exert contrasting, but otherwise concurrent signalling events in bone, the coordinated regu-lation of these opposing effects is not fully under-stood. On page 224 of this issue, Qiu et al. now report an intriguing mechanism for the mutual suppression of PTH and TGF-β signalling in bone. They have identified a PTH-stimulated interaction between the Ser/Thr kinase TβRII and PTH1R, which facilitates their common endocytic internalization, effectively remov-ing both receptors from the cell surface and thereby attenuating the sensitivity of target cells to both ligands6. Thus, PTH suppresses PTH- and TGF-β-mediated signalling in bone and couples bone formation and resorption during the process of bone remodelling.

Several key observations serve as the basis for this proposed model. PTH treatment induces the assembly of a PTH1R–TβRII com-plex that colocalizes with PTH. This complex is initially detected at the plasma membrane,

but is then visualized in endocytic vesicles, indicating that PTH drives internalization of PTH1R–TβRII. Interestingly, PTH-stimulated endocytic trafficking depends on the Ser/Thr kinase activity of TβRII, as expression of a kinase-inactive version of TβRII not only abolishes the interaction between PTH1R and TβRII, but also internalization of that complex. Indeed, Qiu et al. found that TβRII can directly phosphorylate PTH1R at a Ser/Thr cluster in its cytoplasmic tail, a motif that shares strong homology with the Ser/Thr cluster (known as GS domain) of the TGF-β type I receptor (TβRI), another substrate of the TβRII kinase7. Furthermore, gain- and loss-of-function approaches provide compelling evi-dence that TβRII-mediated phosphorylation of PTH1R drives endocytic internalization of the PTH1R–TβRII complex. This previously undescribed biochemical connection between PTH1R and TβRII provides a rational explana-tion for the differential regulation of PTH and TGF-β signalling in biological settings, such as bone homeostasis, that are under dual control of those important physiological modifiers.

Azeddine Atfi is at NSERM UMRS 938, Hôpital St-Antoine, 184 Rue du Faubourg St-Antoine, 75571, Paris, France and Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, 188 Longwood Avenue, Boston, MA 02115, USA. Roland Baron is at Harvard Medical School, Department of Medicine, Endocrine Unit, Massachusetts General Hospital and Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, 188 Longwood Avenue, Boston, MA 02115, USA.e-mail: azeddine.atfi@inserm.fr

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Qiu et al. also explored the consequences of the interaction between PTH1R and TβRII on several downstream aspects of these two signal-ling pathways. TGF-β signalling is initiated by the formation of a heteromeric complex consisting of TβRII and TβRI. Upon ligand binding, TβRII phosphorylates and activates TβRI, which in turn phosphorylates the Smad2/3 intracellular media-tors. Once phosphorylated, Smad2/3 associates with Smad4, and the complexes enter the nucleus where they regulate transcription of TGF-β-responsive genes8,9. Separately, PTH binding to the G protein-coupled PTH1R activates the G protein subunit Gαs. Active Gαs stimulates the production of the second messenger cAMP, which activates protein kinase PKA, which in turn phosphor-ylates multiple transcription factors, including CREB (cAMP responsive element binding pro-tein)3,10. Remarkably, Qiu et al. demonstrated that challenging cells with PTH resulted in decreased

phosphorylation of Smad2 and subsequent asso-ciation with Smad4. PTH also suppressed the activation of transcription in response to TGF-β signalling, as gauged by a gene reporter assay that is widely used to monitor activation of TGF-β signalling11. Thus, the ability of PTH to restrict TGF-β signalling is consistent with the view that PTH can engage TβRII in a complex with PTH1R, which is then internalized by endocyto-sis. Because of the synchronized internalization of PTH1R along with TβRII, such a regulatory mechanism is also expected to have a detrimental effect on PTH signalling. Accordingly, enforced internalization of the PTH1R–TβRII complex through overexpression of TβRII compromises several aspects of PTH signalling, including accumulation of cAMP, phosphorylation of CREB and activation of CREB transcriptional activity. These results clearly indicate that the simultaneous downregulation of PTH and TGF-β

signalling is one important biological con-sequence of endocytic internalization of the PTH1R–TβRII complex (Fig. 1).

Studies in mice with a targeted deletion of TβRII in osteoblasts provided insight into the physiological relevance of the PTH1R–TβRII interaction. As expected from their in vitro studies, Qiu et al. detected an increase in the expression of PTH1R at the cell surface, and consequently, ele-vated PTH signalling, further corroborating the necessity of the interaction between TβRII and PTH1R for increased endocytic internalization of both receptors. Importantly, TβRII deficiency in osteoblasts led to increased bone mass and bone mineral density, with increased production of porous trabecular bone and reduced formation and thickness of cortical bone. This phenotype is reminiscent of that seen in mice bearing a consti-tutively active mutant of PTH1R in osteoblasts12. This finding provides initial evidence that the interplay between TGF-β and PTH receptors also takes place in vivo. Unequivocal support for this hypothesis was provided by the finding that administration of a PTH antagonist (PTH 7-34) to TβRII knockout mice reverses the high bone-mass phenotype and normalizes the trabecular bone volume and the cortical thickness, con-firming that the bone phenotype is an effect of increased PTH signalling.

In an alternative and more robust experimen-tal approach, Qiu et al. investigated the conse-quences of ablating both PTH1R and TβRII in osteoblasts, and found a reversion of the morphological bone changes that are inherent in TβRII deficiency. A remarkable conclusion drawn from these gene interaction studies is that an important component of the TGF-β sig-nalling machinery can also have a crucial role in modulating the anabolic bone response to PTH. Therefore, although the current data primarily implicate PTH as antagonistic to TGF-β signal-ling, a broader exploration of the potential effect of TGF-β on PTH signalling, which might occur by titration of TβRII, could yet reveal additional mechanisms of crosstalk between PTH and TGF-β, with even greater significance for our understanding of bone remodelling.

In addition to this important discovery, Qiu et al. have provided insight both into the crosstalk between PTH and Wnt signalling in bone for-mation13, as well as the importance of TGF-β in bone resorption14. Regarding the former, PTH1R was found to contribute to Wnt-induced bone formation through its interaction with LRP5/6, the co-receptors for activation of canonical Wnt signalling13. Moreover, latent TGF-β stored in

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Figure 1 PTH induces the internalization of TβRII–PTH1R complexes and attenuates PTH and TGF-β signalling. Binding of TGF-β to the TβRI–TβRII complex at the plasma membrane induces phosphorylation and activation of Smad2/3, which then forms a complex with Smad4 and translocates into the nucleus to activate transcription of target genes (blue arrows). PTH binding activates PTH1R to stimulate several downstream effectors, including Gαs, adenylyl cyclase (CA, the enzyme that converts ATP to cAMP, an activator of PKA), PKA, and CREB (red arrows). PTH binding also induces the assembly of a PTH1R–TβRII complex, in which TβRII-mediated phosphorylation of PTH1R induces the subsequent internalization of both PTH1R and TβRII (red arrows). SBE, Smad-binding element; CRE, CREB response element.

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bone matrix was shown to become activated during the osteoclast-driven resorption of bone, creating a gradient of active TGF-β that induces the chemotactic migration of mesenchymal stem cells and osteoblast precursors specifically to pre-viously resorbed sites14. The data from the study by Qiu et al.6 represent an important extension of those previous findings, and provide a significant advance in our understanding of the molecular mechanisms that regulate bone remodelling. Importantly, this study also provides insight into the mechanism by which intermittent PTH administration, an approved anabolic therapy for the treatment of osteoporosis and fractures in the

elderly15, might help to maintain bone mass by interfering with the ability of TGF-β to restrict osteoblast differentiation and maturation. These findings launch a new angle to bone remodel-ling biology, and provide the proof-of-principle for the design and implementation of alternative therapeutic strategies against osteoporosis and other low bone-mass syndromes.

CoMpeting FinAnCiAl inteRestsThe authors declare no competing financial interests.

1. Zaidi, M. Nature Med. 13, 791–801 (2007).2. Potts, J. T. & Gardella, T. J. Ann. NY Acad. Sci. 1117,

196–208 (2007).3. Qin, L., Raggatt, L. J. & Partridge, N. C. Trends

Endocrinol. Metab. 15, 60–65 (2004).

4. Alliston, T., Choy, L., Ducy, P., Karsenty, G. & Derynck, R. EMBO J. 20, 2254–2272 (2001).

5. Alliston, T. N. & Derynck, R. in Skeletal Growth Factors (ed. Canalis, E.) 233–249 (Lippincott Williams & Wilkins Press, Philadelphia (2000).

6. Qiu, T. et al. Nature Cell Biol. 12, 224–234 (2010).7. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F. &

Massague, J. Nature 370, 341–347 (1994).8. Atfi, A. & Baron, R. Sci. Signal. 1, pe33 (2008).9. Shi, Y. & Massague, J. Cell 113, 685–700 (2003).10. Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Nature

Rev. Mol. Cell Biol. 3, 639–650 (2002).11. Shi, Y. et al. Cell 94, 585–594 (1998).12. Calvi, L. M. et al. J. Clin. Invest. 107, 277–286

(2001).13. Wan, M. et al. Genes Dev. 22, 2968–2979 (2008).14. Tang, Y. et al. Nature Med. 15, 757–765 (2009).15. Kousteni, S. & Bilezikian, J. P. Curr. Osteoporos. Rep.

6, 72–76 (2008).

p62, an autophagy hero or culprit?Tor Erik Rusten and Harald Stenmark

The p62 protein recognizes toxic cellular waste, which is then scavenged by a sequestration process known as self‑eating or autophagy. Lack of autophagy leads to accumulation of p62, which is not good for liver cells, as it induces a cellular stress response that leads to disease.

Toxic aggregate-prone proteins, invading microorganisms and damaged organelles are cleared from the cytoplasm by autophagy, a process that involves their engulfment by a double-membrane phagophore that elon-gates and seals to form an autophagosome. Following autophagosome fusion with a lyso-some, the ingested material is degraded by lysosomal enzymes (Fig. 1).

Although originally regarded as a non-selec-tive process, accumulating evidence indicates that autophagy selectively degrades many types of cargo. p62, also known as Sequestosome-1, is one factor that targets specific cargoes for autophagy1. When autophagy is disrupted in several model systems, p62 accumulates with ubiquitin-containing aggregates, a condition reminiscent to that of well-known degenerative disorders. The ubiquitin-positive aggregates that accumulate in autophagy mutants of flies and mice disappear when p62 is depleted2,3. At first glance, p62 thus seems beneficial for the cell, ensuring aggregation and subsequent turnover of potentially harmful proteins by

autophagy. But as usual, too much of a good thing can be bad.

One hint that p62 is not all good came orig-inally from studies by Komatsu et al.2 These authors showed that p62-containing aggre-gates of ubiquitylated proteins accumulated in mouse livers after disruption of autophagy, a phenotype that resembles liver-associated diseases such as hepatocellular carcinoma. In addition, the autophagy-deficient liver cells showed signs of oxidative and electrophilic stress, as shown by transcriptional activa-tion of stress-responsive enzymes. A surpris-ing finding was that p62 removal remedied adverse affects on liver hypertrophy and reversed induction of enzymes produced by oxidative stress. This indicates that p62 accu-mulation itself has a role in the oxidative stress response of the liver.

On page 213, Komatsu et al. explore how excess p62 causes liver damage4, and show that p62-mediated stabilization of a stress regulator leads to deleteriously high production of oxi-dative response genes. The authors investigated the status of Nrf2 (nuclear factor (erythroid-derived 2)-like 2), the master transcriptional regulator of enzymes expressed in response to oxidative and electrophilic cell stress, and found increased nuclear accumulation of Nrf2

in autophagy-deficient cells, accompanied by an overall increase in the expression of Nrf2-regulated genes. Removal of p62 decreased Nrf2 nuclear accumulation, whereas addi-tion of an antioxidant did not. This suggests that p62 positively regulates Nrf2 function independently of the oxidative stress status in the cell.

It seems that p62 can disrupt this system through its interaction with Keap1 (Kelch-like ECH-associated protein 1), a key regulator of Nrf2 that controls Nrf2 transcriptional activ-ity, depending on the stress status of the cell. Under normal conditions, Keap1 acts as an adaptor between the Nrf2 substrate and the Cul3–Rbx ubiquitin ligase complex5 to medi-ate Nrf2 polyubiquitylation and degradation in proteasomes. Under conditions of oxida-tive or electrophilic stress, modifications of redox-sensitive cysteines in Keap1 disrupt its adaptor function and leads to Nrf2 nuclear accumulation and transcription of stress-response enzymes (Fig. 1).

To understand the basis of Nrf2-mediated effects, the authors searched for p62 partners through a proteomics approach and found that it interacts with Keap1. In a heroic effort, the authors used a combination of protein crystallography and biochemistry to map

Tor Erik Rusten and Harald Stenmark are in the Centre for Cancer Biomedicine, University of Oslo and Department of Biochemistry, The Norwegian Radium Hospital Montebello, N-0310, Oslo, Norway.e-mail: h.a.stenmark@medisin.uio.no

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