Lysosomes drive the piecemeal removal of mitochondrial inner membrane.

Mitochondrial membranes define distinct structural and functional compartments. Cristae of the inner mitochondrial membrane (IMM) function as independent bioenergetic units that undergo rapid and transient remodelling, but the significance of this compartmentalized organization is unknown1. Using super-resolution microscopy, here we show that cytosolic IMM vesicles, devoid of outer mitochondrial membrane or mitochondrial matrix, are formed during resting state. These vesicles derived from the IMM (VDIMs) are formed by IMM herniation through pores formed by voltage-dependent anion channel 1 in the outer mitochondrial membrane. Live-cell imaging showed that lysosomes in proximity to mitochondria engulfed the herniating IMM and, aided by the endosomal sorting complex required for transport machinery, led to the formation of VDIMs in a microautophagy-like process, sparing the remainder of the organelle. VDIM formation was enhanced in mitochondria undergoing oxidative stress, suggesting their potential role in maintenance of mitochondrial function. Furthermore, the formation of VDIMs required calcium release by the reactive oxygen species-activated, lysosomal calcium channel, transient receptor potential mucolipin 1, showing an interorganelle communication pathway for maintenance of mitochondrial homeostasis. Thus, IMM compartmentalization could allow for the selective removal of damaged IMM sections via VDIMs, which should protect mitochondria from localized injury. Our findings show a new pathway of intramitochondrial quality control.

The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

Under the Radar: Strategies Used by Helicobacter pylori to Evade Host Responses.

The body depends on its physical barriers and innate and adaptive immune responses to defend against the constant assault of potentially harmful microbes. In turn, successful pathogens have evolved unique mechanisms to adapt to the host environment and manipulate host defenses. Helicobacter pylori (Hp), a human gastric pathogen that is acquired in childhood and persists throughout life, is an example of a bacterium that is very successful at remodeling the host-pathogen interface to promote a long-term persistent infection. Using a combination of secreted virulence factors, immune subversion, and manipulation of cellular mechanisms, Hp can colonize and persist in the hostile environment of the human stomach. Here, we review the most recent and relevant information regarding how this successful pathogen overcomes gastric epithelial host defense responses to facilitate its own survival and establish a chronic infection.

Vitamin D deficiency enhances expression of autophagy-regulating miR-142-3p in mouse and "involved" IBD patient intestinal tissues.

Vitamin D deficiency is an environmental factor involved in the pathogenesis of inflammatory bowel disease (IBD); however, the mechanisms surrounding its role remain unclear. Previous studies conducted in an intestinal epithelial-specific vitamin D receptor (VDR) knockout model suggest that a lack of vitamin D signaling causes a reduction in intestinal autophagy. A potential link between vitamin D deficiency and dysregulated autophagy is microRNA (miR)-142-3p, which suppresses autophagy. In this study, we found that wild-type C57BL/6 mice fed a vitamin D-deficient diet for 5 wk had increased miR-142-3p expression in ileal tissues compared with mice that were fed a matched control diet. Interestingly, there was no difference in expression of key autophagy markers ATG16L1 and LC3II in the ileum whole tissue. However, Paneth cells of vitamin D-deficient mice were morphologically abnormal and had an accumulation of the autophagy adaptor protein p62, which was not present in the total crypt epithelium. These findings suggest that Paneth cells exhibit early markers of autophagy dysregulation within the intestinal epithelium in response to vitamin D deficiency and enhanced miR-142-3p expression. Finally, we demonstrated that treatment-naïve IBD patients with low levels of vitamin D have an increase in miR-142-3p expression in colonic tissues procured from "involved" areas of the disease. Taken together, our findings demonstrate that insufficient vitamin D levels alter expression of autophagy-regulating miR-142-3p in intestinal tissues of mice and patients with IBD, providing insight into the mechanisms by which vitamin D deficiency modulates IBD pathogenesis.NEW & NOTEWORTHY Vitamin D deficiency has a role in IBD pathogenesis, and although the mechanisms surrounding its role remain unclear, it has been suggested that autophagy dysregulation is involved. Here, we show increased ileal expression of autophagy-suppressing miR-142-3p in mice that were fed a vitamin D-deficient diet and in "involved" colonic biopsies from pediatric IBD patients with low vitamin D. miR-142-3p serves as a potential mechanism mediating vitamin D deficiency and reduced autophagy.

MCOLN1/TRPML1 inhibition - a novel strategy used by Helicobacter pylori to escape autophagic killing and antibiotic eradication therapy in vivo.

Inhibition of host macroautophagy/autophagy is one of the strategies used by several intracellular pathogens, including H. pylori, to escape killing. Here we discuss our recent work that revealed the novel mechanism by which the vacuolating cytotoxin A (VacA) produced by H. pylori inhibits lysosomal and autophagic killing. We discovered that VacA impairs the activity of the lysosomal calcium channel MCOLN1/TRPML1 leading to the formation of enlarged, dysfunctional lysosomes and autophagosomes that serve as an intracellular niche, which allows the bacteria to escape eradication therapy.

VacA generates a protective intracellular reservoir for Helicobacter pylori that is eliminated by activation of the lysosomal calcium channel TRPML1.

Helicobacter pylori infection is a proven carcinogen for gastric cancer. Its virulence factor vacuolating cytotoxin A (VacA) promotes more severe disease and gastric colonization. VacA, by an unknown mechanism, usurps lysosomal and autophagy pathways to generate a protected reservoir for H. pylori that confers bacterial survival in vitro. Here, we show the existence of a VacA-generated intracellular niche in vivo that protects the bacteria from antibiotic treatment and leads to infection recrudescence after therapy. Furthermore, we report that VacA targets the lysosomal calcium channel TRPML1 to disrupt endolysosomal trafficking and mediate these effects. Remarkably, H. pylori that lack toxigenic VacA colonize enlarged dysfunctional lysosomes in the gastric epithelium of trpml1-null mice, where they are protected from eradication therapy. Furthermore, a small molecule agonist directed against TRPML1 reversed the toxic effects of VacA on endolysosomal trafficking, culminating in the clearance of intracellular bacteria. These results suggest that TRPML1 may represent a therapeutic target for chronic H. pylori infection.

VacA promotes CagA accumulation in gastric epithelial cells during Helicobacter pylori infection.

Helicobacter pylori (H. pylori) is the causative agent of gastric cancer, making it the only bacterium to be recognized as a Class I carcinogen by the World Health Organization. The virulence factor cytotoxin associated gene A (CagA) is a known oncoprotein that contributes to the development of gastric cancer. The other major virulence factor vacuolating cytotoxin A (VacA), disrupts endolysosomal vesicular trafficking and impairs the autophagy pathway. Studies indicate that there is a functional interplay between these virulence factors by unknown mechanisms. We show that in the absence of VacA, both host-cell autophagy and the proteasome degrade CagA during infection with H. pylori. In the presence of VacA, CagA accumulates in gastric epithelial cells. However, VacA does not affect proteasome function during infection with H. pylori suggesting that VacA-disrupted autophagy is the predominant means by which CagA accumulates. Our studies support a model where in the presence of VacA, CagA accumulates in dysfunctional autophagosomes providing a possible explanation for the functional interplay of VacA and CagA.

Glypican-6 promotes the growth of developing long bones by stimulating Hedgehog signaling.

Autosomal-recessive omodysplasia (OMOD1) is a genetic condition characterized by short stature, shortened limbs, and facial dysmorphism. OMOD1 is caused by loss-of-function mutations of glypican 6 (GPC6). In this study, we show that GPC6-null embryos display most of the abnormalities found in OMOD1 patients and that Hedgehog (Hh) signaling is significantly reduced in the long bones of these embryos. The Hh-stimulatory activity of GPC6 was also observed in cultured cells, where this GPC increased the binding of Hh to Patched 1 (Ptc1). Consistent with this, GPC6 interacts with Hh through its core protein and with Ptc1 through its glycosaminoglycan chains. Hh signaling is triggered at the primary cilium. In the absence of Hh, we observed that GPC6 is localized outside of the cilium but moves into the cilium upon the addition of Hh. We conclude that GPC6 stimulates Hh signaling by binding to Hh and Ptc1 at the cilium and increasing the interaction of the receptor and ligand.

Processing by convertases is required for glypican-3-induced inhibition of Hedgehog signaling.

Glypican-3 (GPC3) is one of the six members of the mammalian glypican family. We have previously reported that GPC3 inhibits Hedgehog (Hh) signaling by competing with Patched (Ptc) for Hh binding. We also showed that GPC3 binds with high affinity to Hh through its core protein, but that it does not interact with Ptc. Several members of the glypican family, including GPC3, are subjected to an endoproteolytic cleavage by the furin-like convertase family of endoproteases. Surprisingly, however, we have found that a mutant GPC3 that cannot be processed by convertases is as potent as wild-type GPC3 in stimulating Wnt activity in hepatocellular carcinoma cell lines and 293T cells and in promoting hepatocellular carcinoma growth. In this study, we show that processing by convertases is essential for GPC3-induced inhibition of Hh signaling. Moreover, we show that a convertase-resistant GPC3 stimulates Hh signaling by increasing the binding of this growth factor to Ptc. Consistent with this, we show that the convertase-resistant mutant binds to both Hh and Ptc through its heparan sulfate (HS) chains. Unexpectedly, we found that the mutant core protein does not bind to Hh. We also report that the convertase-resistant mutant GPC3 carries HS chains with a significantly higher degree of sulfation than those of wild-type GPC3. We propose that the structural changes generated by the lack of cleavage determine a change in the sulfation of the HS chains and that these hypersulfated chains mediate the interaction of the mutant GPC3 with Ptc.

Glypican-3 binds to Frizzled and plays a direct role in the stimulation of canonical Wnt signaling.

Glypican-3 (GPC3) is a proteoglycan that is bound to the cell surface. It is expressed by most hepatocellular carcinomas (HCCs) but not by normal hepatocytes. GPC3 stimulates HCC growth by promoting canonical Wnt signaling. Because glypicans interact with Wnts, it has been proposed that these proteoglycans stimulate signaling by increasing the amount of Wnt at the cell membrane, thus facilitating the interaction of this growth factor with its signaling receptor, Frizzled. However, in this study, we demonstrate that GPC3 plays a more direct role in the stimulation of Wnt signaling. Specifically, we show that, in addition to interacting with Wnt, GPC3 and Frizzled interact directly through the glycosaminoglycan chains of GPC3, indicating that this glypican stimulates the formation of signaling complexes between Wnt and Frizzled. Consistent with this, we show that the binding of Wnt at the cell membrane triggers the endocytosis of a complex that includes Wnt, Frizzled and GPC3. Additional support for our model is provided by the finding that glypican-6 (GPC6) inhibits canonical Wnt signaling, despite the fact that it binds to Wnt at the cell membrane.

The role of glypicans in Hedgehog signaling.

Glypicans (GPCs) are a family of proteoglycans that are bound to the cell surface by a glycosylphosphatidylinositol anchor. Six glypicans have been found in the mammalian genome (GPC1 to GPC6). GPCs regulate several signaling pathways, including the pathway triggered by Hedgehogs (Hhs). This regulation, which could be stimulatory or inhibitory, occurs at the signal reception level. In addition, GPCs have been shown to be involved in the formation of Hh gradients in the imaginal wing disks in Drosophila. In this review we will discuss the role of various glypicans in specific developmental events in the embryo that are regulated by Hh signaling. In addition, we will discuss the mechanism by which loss-of-function GPC3 mutations alter Hh signaling in the Simpson-Golabi-Behmel overgrowth syndrome, and the molecular basis of the GPC5-induced stimulation of Hh signaling and tumor progression in rhabdomyosarcomas.

Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma.

Glypican-3 (GPC3) is a member of the glypican family. Glypicans are proteoglycans that are attached to the cell surface by a glycosyl-phosphatidylinositol anchor. They regulate the signaling activity of several growth factors, including Wnts. This regulation is based on the ability of glypicans to stimulate or inhibit the interaction of these growth factors with their respective signaling receptors. It has been clearly established that whereas GPC3 is expressed by most hepatocellular carcinomas (HCCs), this glypican is not detected in normal and cirrhotic liver, or in benign hepatic lesions. Consequently, immunostaining of liver biopsies for GPC3 is currently being used by clinical pathologists to confirm HCC diagnosis when the malignant nature of the lesion is difficult to establish. In addition to being a marker of HCC, GPC3 plays a role in the progression of the disease. GPC3 promotes the growth of HCC by stimulating canonical Wnt signaling. It has been proposed that this stimulation is based on the ability of GPC3 to increase the binding of Wnt to its signaling receptor, Frizzled. Two therapeutic approaches for HCC that target GPC3 are currently being tested in phase II clinical trials. One of them is based on the use of a humanized GPC3 monoclonal antibody that inhibits the in vivo growth of HCC xenografts by inducing antibody-dependent cellular cytotoxicity. The second approach employs a vaccine that consists of two GPC3-derived peptides that induce cytotoxic T lymphocytes against these peptides. Targeting of GPC3 might offer a new tool for the treatment of HCC.

LRP1 mediates Hedgehog-induced endocytosis of the GPC3-Hedgehog complex.

Glypican-3 (GPC3) is a heparan sulfate (HS) proteoglycan that is bound to the cell membrane through a glycosylphosphatidylinositol link. This glypican regulates embryonic growth by inhibiting the hedgehog (Hh) signaling pathway. GPC3 binds Hh and competes with Patched (Ptc), the Hh receptor, for Hh binding. The interaction of Hh with GPC3 triggers the endocytosis and degradation of the GPC3-Hh complex with the consequent reduction of Hh available for binding to Ptc. Currently, the molecular mechanisms by which the GPC3-Hh complex is internalized remains unknown. Here we show that the low-density-lipoprotein receptor-related protein-1 (LRP1) mediates the Hh-induced endocytosis of the GPC3-Hh complex, and that this endocytosis is necessary for the Hh-inhibitory activity of GPC3. Furthermore, we demonstrate that GPC3 binds through its HS chains to LRP1, and that this interaction causes the removal of GPC3 from the lipid rafts domains.

Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling.

Glypican-5 (GPC5) is one of the six members of the glypican family. It has been previously reported that GPC5 stimulates the proliferation of rhabdomyosarcoma cells. In this study, we show that this stimulatory activity of GPC5 is a result of its ability to promote Hedgehog (Hh) signaling. We have previously shown that GPC3, another member of the glypican family, inhibits Hh signaling by competing with Patched 1 (Ptc1) for Hh binding. Furthermore, we showed that GPC3 binds to Hh through its core protein but not to Ptc1. In this paper, we demonstrate that GPC5 increases the binding of Sonic Hh to Ptc1. We also show that GPC5 binds to both Hh and Ptc1 through its glycosaminoglycan chains and that, unlike GPC3, GPC5 localizes to the primary cilia. Interestingly, we found that the heparan sulfate chains of GPC5 display a significantly higher degree of sulfation than those of GPC3. Based on these results, we propose that GPC5 stimulates Hh signaling by facilitating/stabilizing the interaction between Hh and Ptc1.

Soluble glypican 3 inhibits the growth of hepatocellular carcinoma in vitro and in vivo.

The heterogeneity of the molecular pathology of HCC poses a formidable obstacle to the development of non-cytotoxic therapies. Several pro-tumorigenic signaling pathways can be aberrantly activated in HCC, including those triggered by Wnts. Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan that is overexpressed in most HCCs, promotes the growth of these tumors by stimulating Wnt signaling. Because GPC3 binds with high affinity to Wnts, and its growth-promoting activity requires attachment to the cell membrane, we have hypothesized that a mutated GPC3 lacking the GPI anchoring domain (sGPC3) will block Wnt signaling and inhibit the growth of Wnt-dependent tumors. In addition, because sGPC3 displays heparan sulfate chains, this secreted glypican could also inhibit HCC growth by blocking the activity of other heparin-binding growth factors. To test this hypothesis, HCC cell lines were infected with an sGPC3-expressing lentivirus or virus control, and the effect of sGPC3 on the in vitro and in vivo growth was investigated. In addition, the signaling pathways targeted by sGPC3 were identified. We observed that sGPC3-expressing cells had lower proliferation rate. In addition, sGPC3 significantly inhibited the in vivo growth of the Huh6, HepG2 and Huh7 HCC cell lines. sGPC3 blocked Wnt signaling in Huh6- and Huh7-derived tumors and Erk1/2 and Akt phosphorylation in tumors generated by Huh7 and HepG2 cells, respectively. An anti-angiogenic effect in Huh7 and HepG2-derived tumors was also observed. We conclude that sGPC3 can inhibit HCC tumorigenicity by blocking the activity of several pro-tumorigenic growth factors.

Overgrowth of a mouse model of Simpson-Golabi-Behmel syndrome is partly mediated by Indian hedgehog.

Loss-of-function mutations of Glypican 3 (Gpc3) cause the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and developmental overgrowth is observed in Gpc3-null mice, a mouse model for SGBS. We recently reported that GPC3 inhibits Hedgehog (Hh) signalling by inducing its endocytosis and degradation. Here, we show that the developmental overgrowth observed in Gpc3-null mice is, at least in part, a consequence of the hyperactivation of the Hh pathway. We bred Gpc3-null mice with mice that are Hh signalling-deficient owing to the lack of Indian Hh (Ihh), one of the three mammalian Hhs. We found that the Gpc3-null mice showed a 29.9% overgrowth in an Ihh wild-type background, whereas an Ihh-null background partly rescues the overgrowth caused by the lack of Gpc3 as the double mutants were 19.8% bigger than the Ihh-null mice. Consistent with the role of GPC3 in Hh endocytosis and degradation, the Gpc3-null mice show increased levels of Ihh protein and signalling, but similar levels of Ihh messenger RNA.

The role of glypican-3 in the regulation of body size and cancer.

Glypicans are a family of heparan sulfate proteoglycans whose members are bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Loss-of-function mutations in GPC3, one of the six mammalian glypicans, causes the Simson-Golabi-Behmel Syndrome. This is a disorder characterized by pre- and post-natal overgrowth, a broad spectrum of visceral and skeletal abnormalities, and an increased risk for the development of embryonic tumors. GPC3-null mice also display significant overgrowth. We have recently reported that GPC3 acts as a negative regulator of Hedgehog signaling during development, and that the overgrowth caused by the lack of functional GPC3 is due, at least in part, to the hyperactivation of Hedgehog signaling. Here we discuss the rationale that led us to hypothesize that GPC3 could be a negative regulator of Hedgehog signaling, and speculate about the implications of our discovery regarding the role of GPC3 in some cancer types. We also discuss our recent results of experiments that investigated the role of the core protein, the heparan sulfate chains, and the GPI anchor in GPC3 function. Finally, we propose an explanation for the tissue-specific function of GPC3.

Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding.

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.

Glypicans.

SUMMARY: Glypicans are heparan sulfate proteoglycans that are bound to the outer surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor. Homologs of glypicans are found throughout the Eumetazoa. There are six family members in mammals (GPC1 to GPC6). Glypicans can be released from the cell surface by a lipase called Notum, and most of them are subjected to endoproteolytic cleavage by furin-like convertases. In vivo evidence published so far indicates that the main function of membrane-attached glypicans is to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors and bone morphogenetic proteins (BMPs). Depending on the context, glypicans may have a stimulatory or inhibitory activity on signaling. In the case of Wnt, it has been proposed that the stimulatory mechanism is based on the ability of glypicans to facilitate and/or stabilize the interaction of Wnts with their signaling receptors, the Frizzled proteins. On the other hand, GPC3 has recently been reported to inhibit Hedgehog protein signaling during development by competing with Patched, the Hedgehog receptor, for Hedgehog binding. Surprisingly, the regulatory activity of glypicans in the Wnt, Hedgehog and BMP signaling pathways is only partially dependent on the heparan sulfate chains.

Immunohistochemical study of glypican 3 in thyroid cancer.

In 123 patients with thyroid cancer, expression of glypican 3 (GPC3) was immunohistochemically investigated in tissue samples and the biological significance of GPC3 in thyroid cancer was examined. GPC3 was scarcely expressed in the normal thyroid gland, but was dramatically enhanced in certain types of cancers: 100% in follicular carcinoma (20/20 cases) and 70% in papillary carcinoma (48/69 cases). Expression of GPC3 in follicular carcinoma was significantly higher than that of follicular adenoma (p < 0.0019). In contrast, GPC 3 was not expressed in 17 cases of anaplastic carcinoma. A high expression of GPC3 mRNA was confirmed in cancer lesions, which were strongly positive for immunohistochemical staining. In 69 cases of papillary carcinoma, GPC3 was expressed at an early stage, suggesting that GPC3 expression in thyroid cancer is an early event in developing papillary carcinoma. Further studies are required to determine biological functions and molecular mechanisms underlying the upregulation of GPC3 in thyroid cancer.