Portal de Periódicos da CAPES

Regulated intramembrane proteolysis of megalin: Linking urinary protein and gene regulation in proximal tubule?

2006; Elsevier BV; Volume: 69; Issue: 10 Linguagem: Inglês

10.1038/sj.ki.5000298

1523-1755

Daniel Biemesderfer,

Cerebrovascular and genetic disorders

Regulated intramembrane proteolysis (RIP) represents an evolutionarily conserved process linking receptor function with transcriptional regulation. Best characterized by the Notch signaling pathway, RIP involves regulated ectodomain shedding followed by γ-secretase-mediated release of the C-terminal, cytosolic domain. The C-terminus in turn translocates to the nucleus where it interacts with other proteins to regulate expression of specific genes. Recent studies in our laboratory have shown that megalin, a scavenger receptor in proximal tubule, is subjected to RIP in a manner very similar to that of Notch. We showed that megalin in subjected to protein kinase C-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-associated C-terminal fragment (MCTF). The MCTF in turn forms the substrate for γ-secretase. These data implicate megalin as a central element of a Notch-like signaling pathway linking protein reabsorption and gene regulation in proximal tubule. The likelihood that megalin processing plays an important role in the progression of proteinuric kidney disease is discussed. Regulated intramembrane proteolysis (RIP) represents an evolutionarily conserved process linking receptor function with transcriptional regulation. Best characterized by the Notch signaling pathway, RIP involves regulated ectodomain shedding followed by γ-secretase-mediated release of the C-terminal, cytosolic domain. The C-terminus in turn translocates to the nucleus where it interacts with other proteins to regulate expression of specific genes. Recent studies in our laboratory have shown that megalin, a scavenger receptor in proximal tubule, is subjected to RIP in a manner very similar to that of Notch. We showed that megalin in subjected to protein kinase C-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-associated C-terminal fragment (MCTF). The MCTF in turn forms the substrate for γ-secretase. These data implicate megalin as a central element of a Notch-like signaling pathway linking protein reabsorption and gene regulation in proximal tubule. The likelihood that megalin processing plays an important role in the progression of proteinuric kidney disease is discussed. Elevated urinary protein is one of the most significant predictors of renal disease and controlling proteinuria in the patient with proteinuric kidney disease is paramount for the physician. Increasing evidence from both animal and cell culture models show that chronic proteinuria not only acts as an index of renal pathology but also that elevated protein is toxic to the renal tubular epithelium. Although the molecular mechanisms of this toxicity are largely unknown, experimental evidence indicates that albumin can activate transcription of both NF-κB-dependent and NF-κB-independent genes leading to increased secretion of both cytokines and chemokines by proximal tubule cells. It has been suggested that this change in phenotype of the proximal tubule in turn leads to the inflamed and fibrotic states seen in the proteinuric kidney. For a recent review, see Gekle.1.Gekle M. Renal tubule albumin transport.Annu Rev Physiol. 2005; 67: 573-594Crossref PubMed Scopus (218) Google Scholar Understanding in detail the molecular mechanisms of tubular protein reabsorption and how this process may regulate gene expression in the proximal tubule is an important goal for nephrologists. The proximal tubule reabsorbs filtered protein from the urine largely by clathrin-dependent, receptor-mediated endo-cytosis. It is clear from biochemical and gene knockout studies that the scavenger receptor megalin performs a central role in renal protein reabsorption.2.Christensen E.I. Birn H. Megalin and cubilin: multifunctional endocytic receptors.Nat Rev Mol Cell Biol. 2002; 3: 256-266Crossref PubMed Scopus (650) Google Scholar A member of the low-density lipoprotein-receptor gene family, megalin was first discovered and identified as the Heymann nephritis autoantigen by Kerjaschki and Farquhar in 1982.3.Kerjaschki D. Farquhar M.G. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border.Proc Natl Acad Sci USA. 1982; 79: 5557-5581Crossref PubMed Scopus (406) Google Scholar Originally called gp330, megalin was shown to be highly expressed in the clathrin-coated pit region of the renal brush border and, because of its subcellular location, predicted to be involved in endocytosis. Subsequent studies have identified an impressive list of ligands that bind megalin in vitro and include albumin, vitamin-binding proteins, carrier proteins, lipoproteins, hormones such as insulin, drugs and toxins, enzymes, and others such as Ca2+ and receptor-associated protein. For an excellent review, see Christensen and Birn.2.Christensen E.I. Birn H. Megalin and cubilin: multifunctional endocytic receptors.Nat Rev Mol Cell Biol. 2002; 3: 256-266Crossref PubMed Scopus (650) Google Scholar Although the physiologic importance of megalin's binding to many of the ligands described in the literature is not known, it is generally accepted that megalin functions as a scavenger receptor in the proximal tubule. Recently, new data from our laboratory showing that megalin is subjected to a process called regulated intramembrane proteolysis (RIP) provide a working model linking megalin-dependent receptor-mediated endocytosis with cell signaling events. These findings may also help to explain at the molecular level the link between chronic proteinuria and the pathology seen clinically in patients with proteinuric renal diseases. As part of their studies of cholesterol metabolism, Brown and Goldstein and co-workers discovered that a receptor involved in cholesterol metabolism, called the sterol regulatory element binding protein, was subjected to a series of regulated proteolytic cleavages. Specific proteolytic fragments of sterol regulatory element binding produced during this process were shown to be translocated to the nucleus, resulting in transcriptional regulation of specific genes involved in both fatty acid and cholesterol metabolism4.Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2978) Google Scholar). RIP of sterol regulatory element binding is now known to represent an evolutionarily conserved process that acts on numerous receptors and link receptor-mediated endocytosis with intracellular signaling events. Among the growing list of receptors now known to be subjected to RIP, Notch and the amyloid precursor protein (APP) are probably the best studied. Notch, a 300 kDa single membrane pass receptor, was first described in Drosophila melanogaster and shown to be necessary for neural development in that species. It is now clear that Notch is part of a signaling pathway that determines cell fate in many tissues in both vertebrates and invertebrates alike. Proteolytic processing of Notch appears to be similar, if not identical, to processing described for the APP. Understanding the proteolytic processing steps of APP has become the focus of many studies because increasing evidence indicates that anomalies in APP processing by α, β, and γ secretases result in the production of pathogenic fragments, known as the amyloid β peptides, which have been linked to the onset and progression of Alzheimer's disease. An overview of the relevant features of the RIP of Notch is shown in Figure 1 and is summarized below. The initial step (2) in RIP is ectodomain shedding.5.Schroeter E.H. Kisslinger J.A. Kopan R. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.Nature. 1998; 393: 382-386Crossref PubMed Scopus (1358) Google Scholar The shedding activity is protein kinase C-regulated and frequently occurs as multiple steps involving distinct proteases for each step. The role of ligand binding (1) for RIP seems to be quite variable. In some instances, for example in the case of Notch, ligand binding is required for ectodomain shedding to occur. However, for other receptors, including APP, the role of ligand binding is unclear and ectodomain shedding along with all subsequent processing steps seem to be largely constituitive. Ectodomain shedding is carried out by one or more members of the metalloprotease-disintegrin family of proteases, also called ADAMs (a disintegrin and metalloprotease domain). ADAM17, also called tumor necrosis factor-α-converting enzyme and ADAM10 (Kuzbanian) appear to be important enzymes for ectodomain shedding of both Notch and APP.6.Hartmann D. Tournoy J. Saftig P. et al.Implication of APP secretases in notch signaling.J Mol Neurosci. 2001; 17: 171-181Crossref PubMed Scopus (64) Google Scholar Ectodomain shedding of Notch yields characteristic membrane-associated C-terminal fragments (referred to as NCTF in Figure 1). The NCTF in turn forms the substrate for γ-secretase activity (3) acting at a site within the transmembrane domain. The γ-secretase cleavage site has been identified by N-terminal sequencing for both Notch and APP, and shown to occur immediately before a valine residue located 3–4 amino acids from the putative membrane/cytosolic boundary.7.Xia W. Wolfe M.S. Intramembrane proteolysis by presenilin and presenilin-like proteases.J Cell Sci. 2003; 116: 2839-2844Crossref PubMed Scopus (78) Google Scholar γ-Secretase activity is mediated by a protein complex that includes the presenilins (1 and 2), which appear to mediate the proteolytic activity, as well as 'accessory' proteins pen-2, aph-1, and nicastrin whose functions are largely unknown. New studies suggest that additional posttranslational steps may be required for the γ-secretase cleavage to occur. Gupta-Rossi and co-workers have shown that monoubiquitination and endocytosis of the NCTF are both required prior to cleavage by γ-secretase.8.Gupta-Rossi N. Six E. LeBail O. et al.Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor.J Cell Biol. 2004; 166: 73-83Crossref PubMed Scopus (182) Google Scholar Once cleaved, the soluble C-terminal domain is trafficked to the nucleus (4), where it interacts with transcription factors and regulates expression of specific genes. The intracellular domain of Notch interacts with the CSL (CSL stands for CBF1, Su(H), Lag-1) family of DNA-binding proteins (5). In 2002, members of Joachim Herz' laboratory published a study showing that a protein closely related to megalin called low density lipoprotein receptor related protein is also subjected to RIP.9.May P. Reddy Y.K. Herz J. Proteolytic processing of low density lipoprotein receptor-related protein mediates regulated release of its intracellular domain.J Biol Chem. 2002; 277: 18736-18743Crossref PubMed Scopus (260) Google Scholar In a subsequent review discussing the relationship between endocytosis and cellular signaling of lipoprotein receptors, May and Herz suggested that megalin may also be subjected to RIP and might be involved in regulating genes of the vitamin D metabolic pathway in the proximal tubule.10.May P. Bock H.H. Herz J. Integration of endocytosis and signal transduction by lipoprotein receptors.Sci STKE. 2003; 2003: PE12PubMed Google Scholar This idea was founded largely on work by Willnow and co-workers, who had earlier shown megalin is required for the uptake and activation of 25-(OH) vitamin D3 by the proximal tubule.11.Nykjaer A. Dragun D. Walther D. et al.An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3.Cell. 1999; 96: 507-515Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar Willnow's group also identified, using the yeast-two-hybrid technique, a protein called MegBP (for megalin-binding protein), which interacted with both the C-terminal cytosolic domain of megalin and with SKI-interacting protein, a transcriptional regulator and co-activator of the vitamin D receptor.12.Petersen H.H. Hilpert J. Militz D. et al.Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule.J Cell Sci. 2003; 116: 453-461Crossref PubMed Scopus (42) Google Scholar With this in mind we began studies designed to look for RIP of megalin in kidney. Interestingly, we noticed conservation of the predicted critical valine residue (the γ-secretase cleavage site) in the transmembrane domain of megalin. Initially, we sought to determine if significant levels of γ-secretase activity, as well as presenilin protein, are expressed in renal brush border. Figure 2 shows that immunocytochemical studies were able to localize presenilin-1 to the brush border as well as to the endocytic apparatus of proximal tubule cells. The localization of presenilin to the renal brush border was supported biochemically by the findings that high levels of presenilin-1 protein and γ-secretase activity were enriched in renal brush border membrane vesicles.13.Zou Z. Chung B. Nguyen T. et al.Linking receptor-mediated endocytosis and cell signaling: Evidence for regulated intramembrane proteolysis of megalin in proximal tubule.J Biol Chem. 2004; 279: 34302-34310Crossref PubMed Scopus (141) Google Scholar Therefore, the subcellular location of presenilin-1, and thus γ-secretase, in proximal tubule significantly overlaps that described for megalin. In order to perform more detailed biochemical studies and look for Notch-like processing of megalin, we utilized a well-established cell line derived from opossum proximal tubule called OKP. OKP cells express significant levels of megalin and have been used by several laboratories to study the interrelationship between protein absorption and gene expression in proximal tubule. Studies from Gekle's laboratory have established megalin to be the primary receptor for receptor-mediated endocytosis of albumin in this cell line.14.Zhai X.Y. Nielsen R. Birn H. et al.Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.Kidney Int. 2000; 58: 1523-1533Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar More importantly, endocytosis of albumin, again presumably by a megalin-dependent pathway, is now known to result in the specific activation of several genes in cultured proximal tubule cells including fractalkine (CX3CL1),15.Donadelli R. Zanchi C. Morigi M. et al.Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappaB- and p38 mitogen-activated protein kinase-dependent pathways.J Am Soc Nephrol. 2003; 14: 2436-2446Crossref PubMed Scopus (114) Google Scholar RANTES (regulated upon activation, normal T cell expressed and secreted) 16.Zoja C. Donadelli R. Colleoni S. et al.Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation.Kidney Int. 1998; 53: 1608-1615Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar and the brush border Na+/H+ exchanger, NHE3.17.Klisic J. Zhang J. Nief V. et al.Albumin regulates the Na+/H+ exchanger 3 in OKP cells.J Am Soc Nephrol. 2003; 14: 3008-3016Crossref PubMed Scopus (38) Google Scholar Our laboratory has cloned the C-terminal region of megalin from OKP cells and has developed both monoclonal and polyclonal antibodies to this region of the receptor. These reagents have been applied to studies of OKP cells and used successfully to study the biogenesis of megalin and especially its proteolytic processing in this cell line. We discovered that, like Notch, APP and low density lipoprotein receptor related protein, the ectodomain of megalin is shed (Figure 3, (2)) in OKP cells by a protein kinase C-regulated, metalloprotease-mediated activity.13.Zou Z. Chung B. Nguyen T. et al.Linking receptor-mediated endocytosis and cell signaling: Evidence for regulated intramembrane proteolysis of megalin in proximal tubule.J Biol Chem. 2004; 279: 34302-34310Crossref PubMed Scopus (141) Google Scholar Ectodomain shedding produced a 40–45 kDa membrane bound, C-terminal fragment of the receptor, which we refer to as the megalin C-terminal fragment or MCTF (Figure 3). Importantly, the MCTF is the same size as a major C-terminal fragment of megalin found in kidney and is similar to C-terminal fragments produced when other receptors are similarly subjected to ectodomain shedding. Although the exact identity of the megalin sheddase(s) and the cleavage site are unknown, the mechanism seems to be similar, if not identical, to that described for other receptors subjected to RIP. We found that ectodomain shedding of megalin had both constituitive and ligand-dependent (1) components. Specific megalin ligands, such as vitamin D-binding protein, activated the shedding process. In these experiments, the MCTF produced by ectodomain shedding formed the substrate for γ-secretase activity (3). The half-life of the megalin intracellular domain thus formed is very short, and we have not been able to follow its subcellular trafficking (4) (i.e. to the nucleus). This again is consistent with data derived from studies of other receptors. For example, the γ-secretase product of endogenous Notch, the Notch intracellular domain, is not detectable and studies that have localized the Notch intracellular domain to the nucleus have done so by overexpressing the Notch gene in cultured cells. In agreement with the prediction of May and Herz, we conclude from these studies that key elements of a Notch-like signaling pathway, using megalin as a novel substrate, exists in the proximal tubule. The downstream events of this pathway shown in Figure 3 such as targeting of the megalin intracellular domain to the nucleus and gene regulation are predictions based on other similar pathways such as Notch. Our data show that RIP of megalin can be activated by vitamin D-binding protein and are consistent with the idea that this pathway regulates genes involved in vitamin D metabolism in the proximal tubule. However, regulation of a specific set of genes involved in vitamin D metabolism would be difficult to reconcile if megalin is in fact a multiligand, scavenger receptor. Therefore, an important question that will need to be addressed is: 'If the megalin signaling pathway is activated by ligands, do all megalin ligands activate processing equally and activate the same genes?' The fact that megalin is expressed in the brush border of the proximal tubule means that it is in direct contact with the glomerular ultrafiltrate. Another intriguing role for megalin as a signaling molecule in proximal tubule may be that it acts as a 'sensor' of urinary protein filtered by the glomerulus. In this case, megalin processing might regulate brush border gene products involved in brush border activity, and thus directly influence the absorptive capacity of proximal tubule cells. The interrelationship between endocytosis and cell signaling is a well-established process.18.Di Fiore P.P. De Camilli P. Endocytosis and signaling; an inseparable partnership.Cell. 2001; 106: 1-4Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 19.Cavalli V. Corti M. Gruenberg J. Endocytosis and signaling cascades: a close encounter.FEBS Lett. 2001; 498: 190-196Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar In proximal tubule, a model of endocytosis-regulated protein expression, possibly at the transcriptional level, is supported by gene knockout studies. Jentsch's group has developed a knockout the brush border endosomal Cl- channel, ClC-5. In addition to inhibiting receptor-mediated endocytosis in proximal tubule, the loss of ClC-5 also inhibits megalin protein expression.20.Piwon N. Gunther W. Schwake M. et al.ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease.Nature. 2000; 408: 369-373Crossref PubMed Scopus (482) Google Scholar More dramatically, megalin knock animals exhibit poorly developed brush borders. These animals have severe proteinuria as might be expected from megalin loss of function. However, the proximal tubules of these animals also exhibit a markedly reduced endocytic pathway suggesting that some aspect of megalin function regulates expression of other proteins involved in endocytosis. The appearance of coated pits, endosomes, and dense apical tubules are generally reduced in the absence of megalin. The increased appearance of specific proteins, for example vitamin D-binding protein, in the urine of the megalin -/- animals has been offered as evidence that megalin is a receptor for these proteins. However, it is difficult to reconcile if the proteinuria seen in these animals results from loss of the receptor function of megalin or from the generalized reduction of the endocytic pathway itself. It is tempting to speculate that the proteinuria seen in the megalin-/- animals might result from the loss of megalin function as a signaling molecule rather than as a receptor per se. Additional studies will be required to understand how the receptor and signaling functions of megalin interact and relate to one another. What role might megalin signaling play in proteinuric kidney disease? It is easy to imagine that a megalin-dependent signaling pathway in proximal tubule would be dramatically impacted during states of chronic proteinuria. Knowledge of the downstream signaling events of this pathway, especially with regard to the genes that might be regulated, may have important consequences for our understanding of the onset and progression renal disease. The albumin-dependent changes in gene expression seen in cultured proximal tubule cells may be mediated directly through megalin's signaling function. This function could also explain the change in phenotype of proximal tubules during proteinuric kidney disease, and may participate in the progressive renal damage seen in the nephrotic state. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54933 and by a Scherbenske Grant from the American Society of Nephrology.