Regulation of muscle hypertrophy through granulin: Relayed communication among mesenchymal progenitors, macrophages, and satellite cells.

Skeletal muscles exert remarkable regenerative or adaptive capacities in response to injuries or mechanical loads. However, the cellular networks underlying muscle adaptation are poorly understood compared to those underlying muscle regeneration. We employed single-cell RNA sequencing to investigate the gene expression patterns and cellular networks activated in overloaded muscles and compared these results with those observed in regenerating muscles. The cellular composition of the 4-day overloaded muscle, when macrophage infiltration peaked, closely resembled that of the 10-day regenerating muscle. In addition to the mesenchymal progenitor-muscle satellite cell (MuSC) axis, interactome analyses or targeted depletion experiments revealed communications between mesenchymal progenitors-macrophages and macrophages-MuSCs. Furthermore, granulin, a macrophage-derived factor, inhibited MuSC differentiation, and Granulin-knockout mice exhibited blunted muscle hypertrophy due to the premature differentiation of overloaded MuSCs. These findings reveal the critical role of granulin through the relayed communications of mesenchymal progenitors, macrophages, and MuSCs in facilitating efficient muscle hypertrophy.

Structural analysis of a U-superfamily conotoxin containing a mini-granulin fold: Insights into key features that distinguish between the ICK and granulin folds.

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold; however, AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms.

Granulin in renal tubular epithelia is associated with interstitial inflammation and activates the TLR9-IFN-α pathway in lupus nephritis.

OBJECTIVE: This study aimed to investigate the possible role of granulin (GRN) in activating the TLR9-IFN-α pathway in renal tubular epithelial cells (RTECs) and explore clues that RTECs regulate the micro-environment of inflammatory response in lupus nephritis (LN). METHODS: Renal sections from 57 LN patients and 30 non-LN patients were sampled for histological study, and GRN overexpression RTECs were applied for cytological study. RESULTS: In the histological study, GRN is highly expressed in LN RTECs with tubulointerstitial inflammation (TII) and well co-localized with TLR9. ROC analysis suggested a potential relationship between GRN expression in RTECs and therapeutic response. Moreover, IFN-α also highly expressed in LN RTECs with TII, and the intensity of IFN-α is positively correlated with the co-localization intensity of GRN and TLR9. In the cytological study, LN serum, especially serum from LN with TII, activates the expression of TLR9 in RTECs, and GRN engages the interaction of TLR9 to activate the expression of IFN-α in RTECs. While TLR9 inhibitors can suppress the expression of IFN-α in RTECs, the degree of inhibition is dose-dependent. CONCLUSION: The expression of GRN in RTECs is associated with interstitial inflammation and therapeutic response. GRN may mediate the activation of the TLR9-IFN-α pathway in RTECs and involve in the micro-environment of inflammatory response in LN.

Progranulin haploinsufficiency mediates cytoplasmic TDP-43 aggregation with lysosomal abnormalities in human microglia.

BACKGROUND: Progranulin (PGRN) haploinsufficiency due to progranulin gene (GRN) variants can cause frontotemporal dementia (FTD) with aberrant TAR DNA-binding protein 43 (TDP-43) accumulation. Despite microglial burden with TDP-43-related pathophysiology, direct microglial TDP-43 pathology has not been clarified yet, only emphasized in neuronal pathology. Thus, the objective of this study was to investigate TDP-43 pathology in microglia of patients with PGRN haploinsufficiency. METHODS: To design a human microglial cell model with PGRN haploinsufficiency, monocyte-derived microglia (iMGs) were generated from FTD-GRN patients carrying pathogenic or likely pathogenic variants (p.M1? and p.W147*) and three healthy controls. RESULTS: iMGs from FTD-GRN patients with PGRN deficiency exhibited severe neuroinflammation phenotype and failure to maintain their homeostatic molecular signatures, along with impaired phagocytosis. In FTD-GRN patients-derived iMGs, significant cytoplasmic TDP-43 aggregation and accumulation of lipid droplets with profound lysosomal abnormalities were observed. These pathomechanisms were mediated by complement C1q activation and upregulation of pro-inflammatory cytokines. CONCLUSIONS: Our study provides considerable cellular and molecular evidence that loss-of-function variants of GRN in human microglia can cause microglial dysfunction with abnormal TDP-43 aggregation induced by inflammatory milieu as well as the impaired lysosome. Elucidating the role of microglial TDP-43 pathology in intensifying neuroinflammation in individuals with FTD due to PGRN deficiency and examining consequential effects on microglial dysfunction might yield novel insights into the mechanisms underlying FTD and neurodegenerative disorders.

The progranulin cleavage product granulin 3 exerts a dominant negative effect on animal fitness.

Progranulin is an evolutionarily conserved protein that has been implicated in human neurodevelopmental and neurodegenerative diseases. Human progranulin is comprised of multiple cysteine-rich, biologically active granulin peptides. Granulin peptides accumulate with age and stress, however their functional contributions relative to full-length progranulin remain unclear. To address this, we generated C. elegans strains that produced quantifiable levels of both full-length progranulin/PGRN-1 protein and cleaved granulin peptide. Using these strains, we demonstrated that even in the presence of intact PGRN-1, granulin peptides suppressed the activity of the lysosomal aspartyl protease activity, ASP-3/CTSD. Granulin peptides were also dominant over PGRN-1 in compromising animal fitness as measured by progress through development and stress response. Finally, the degradation of human TDP-43 was impaired when the granulin to PGRN-1 ratio was increased, representing a disease-relevant downstream impact of impaired lysosomal function. In summary, these studies suggest that not only absolute progranulin levels, but also the balance between full-length progranulin and its cleavage products, is important in regulating lysosomal biology. Given its relevance in human disease, this suggests that the processing of progranulin into granulins should be considered as part of disease pathobiology and may represent a site of therapeutic intervention.

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors.

αD-conotoxins are 11 kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein (Ls-AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls-AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

Clonorchis sinensis granulin promotes malignant transformation of human intrahepatic biliary epithelial cells through interaction with M2 macrophages via regulation of STAT3 phosphorylation and the MEK/ERK pathway.

BACKGROUND: Clonorchis sinensis granulin (CsGRN), a component of the excretory/secretory products of this species, is a multifunctional growth factor that can promote the metastasis of cholangiocarcinoma cells. However, the effect of CsGRN on human intrahepatic biliary epithelial cells (HIBECs) is unclear. Here, we investigated the effect of CsGRN on the malignant transformation of HIBECs and its possible underlying mechanism. METHODS: The malignant transformation phenotypes of HIBECs after CsGRN treatment were estimated by EdU-488 incorporation assay, colony formation assay, wound-healing assay, Transwell assay and western blot. The biliary damage of CsGRN-treated mice was detected by western blot, immunohistochemical staining and hematoxylin and eosin staining. The phenotypes of the macrophages [human monocytic leukemia cell line (THP-1)] were analyzed by flow cytometry, immunofluorescence and immunohistochemistry, both in vitro and in vivo. A co-culture system was developed to explore the interaction between THP-1 and HIBECs in CsGRN-containing medium. Enzyme-linked immunosorbent assay and western blot were used to detected the activation of interleukin 6 (IL-6), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. An inhibitor of the MEK/ERK pathway, PD98059, was used to determine whether this pathway is involved in CsGRN-mediated cell interactions as well as in STAT3 phosphorylation and malignant transformation of HIBECs. RESULTS: Excessive hyperplasia and abnormal proliferation of HIBECs, enhanced secretion of hepatic pro-inflammatory cytokines and chemokines, as well as biliary damage, were observed in vitro and in vivo after treatment with CsGRN. The expression of the markers of M2 macrophages significantly increased in CsGRN-treated THP-1 cells and biliary duct tissues compared with the controls. Moreover, following treatment with CsGRN, the HIBECs underwent malignant transformation in the THP-1-HIBECs co-culture group. In addition, high expression of IL-6 was observed in the CsGRN-treated co-culture media, which activated the phosphorylation of STAT3, JAK2, MEK and ERK. However, treatment with an inhibitor of the MEK/ERK pathway, PD98059, decreased expression of p-STAT3 in CsGRN-treated HIBECs and further repressed the malignant transformation of HIBECs. CONCLUSIONS: Our results demonstrated that, by inducing the M2-type polarization of macrophages and activating the IL-6/JAK2/STAT3 and MEK/ERK pathways in HIBECs, CsGRN promoted the malignant transformation of the latter.

SWAP70 Overexpression Protects Against Pathological Cardiac Hypertrophy in a TAK1-Dependent Manner.

Background Pathological cardiac hypertrophy is regarded as a critical precursor and independent risk factor of heart failure, and its inhibition prevents the progression of heart failure. Switch-associated protein 70 (SWAP70) is confirmed important in immunoregulation, cell maturation, and cell transformation. However, its role in pathological cardiac hypertrophy remains unclear. Methods and Results The effects of SWAP70 on pathological cardiac hypertrophy were investigated in Swap70 knockout mice and Swap70 overexpression/knockdown cardiomyocytes. Bioinformatic analysis combined with multiple molecular biological methodologies were adopted to elucidate the mechanisms underlying the effects of SWAP70 on pathological cardiac hypertrophy. Results showed that SWAP70 protein levels were significantly increased in failing human heart tissues, experimental transverse aortic constriction-induced mouse hypertrophic hearts, and phenylephrine-stimulated isolated primary cardiomyocytes. Intriguingly, phenylephrine treatment decreased the lysosomal degradation of SWAP70 by disrupting the interaction of SWAP70 with granulin precursor. In vitro and in vivo experiments revealed that Swap70 knockdown/knockout accelerated the progression of pathological cardiac hypertrophy, while Swap70 overexpression restrained the cardiomyocyte hypertrophy. SWAP70 restrained the binding of transforming growth factor ß-activated kinase 1 (TAK1) and TAK1 binding protein 1, thus blocking the phosphorylation of TAK1 and downstream c-Jun N-terminal kinase/P38 signaling. TAK1 interacted with the N-terminals (1-192) of SWAP70. Swap70 (193-585) overexpression failed to inhibit cardiac hypertrophy when the TAK1-SWAP70 interaction was disrupted. Either inhibiting the phosphorylation or suppressing the expression of TAK1 rescued the exaggerated cardiac hypertrophy induced by Swap70 knockdown. Conclusions SWAP70 suppressed the progression of cardiac hypertrophy, possibly by inhibiting the mitogen-activated protein kinases signaling pathway in a TAK1-dependent manner, and lysosomes are involved in the regulation of SWAP70 expression level.

Interaction with ERp57 is required for progranulin protection against Type 2 Gaucher disease.

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates ß-glucosylceramide (ß-GlcCer). We reported progranulin (PGRN), a secretary growth factor-like molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and ß-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD.

A variant in GRN of Spanish origin presenting with heterogeneous phenotypes.

INTRODUCTION: The variant c.1414-1G>T in the GRN gene has previously been reported as probably pathogenic in subjects of Hispanic origin in the American continent. METHODS: We report 5 families of Spanish origin carrying this variant, including the clinical, neuroimaging, and laboratory findings. RESULTS: Phenotypes were strikingly different, including cases presenting with behavioral variant frontotemporal dementia, semantic variant primary progressive aphasia, rapidly progressive motor neuron disease (pathologically documented), and tremor-dominant parkinsonism. Retinal degeneration has been found in homozygous carriers only. Ex vivo splicing assays confirmed that the mutation c.1414-1G>T affects the splicing of the exon, causing a loss of 20 amino acids in exon 11. CONCLUSIONS: We conclude that variant c.1414-1G>T of the GRN gene is pathogenic, can lead to a variety of clinical presentations and to gene dosage effect, and probably has a Spanish founder effect.

Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

Structural brain splitting is a hallmark of Granulin-related frontotemporal dementia.

Frontotemporal dementia associated with granulin (GRN) mutations presents asymmetric brain atrophy. We applied a Minimum Spanning Tree plus an Efficiency Cost Optimization approach to cortical thickness data in order to test whether graph theory measures could identify global or local impairment of connectivity in the presymptomatic phase of pathology, where other techniques failed in demonstrating changes. We included 52 symptomatic GRN mutation carriers (SC), 161 presymptomatic GRN mutation carriers (PSC) and 341 non-carriers relatives from the Genetic Frontotemporal dementia research Initiative cohort. Group differences of global, nodal and edge connectivity in (Minimum Spanning Tree plus an Efficiency Cost Optimization) graph were tested via Structural Equation Models. Global graph perturbation was selectively impaired in SC compared to non-carriers, with no changes in PSC. At the local level, only SC exhibited perturbation of frontotemporal nodes, but edge connectivity revealed a characteristic pattern of interhemispheric disconnection, involving homologous parietal regions, in PSC. Our results suggest that GRN-related frontotemporal dementia resembles a disconnection syndrome, with interhemispheric disconnection between parietal regions in presymptomatic phases that progresses to frontotemporal areas as symptoms emerge.

Differential regulation of progranulin derived granulin peptides.

BACKGROUND: Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN is comprised of 7.5 granulin repeats and is processed into individual granulin peptides in the lysosome. However, very little is known about the levels and regulations of individual granulin peptides due to the lack of specific antibodies. RESULTS: Here we report the generation and characterization of antibodies specific to each granulin peptide. We found that the levels of granulins C, E and F are regulated differently  compared to granulins A and B in various tissues. The levels of PGRN and granulin peptides vary in different brain regions and the ratio between granulins and PGRN is highest in the cortical region in the adult male mouse brain. Granulin-A is localized in the lysosome in both neurons and microglia and its levels in microglia increase under pathological conditions. Interestingly,  the levels of granulin A in microglia change correspondingly with PGRN in response to stroke but not demyelination. Furthermore, deficiency of lysosomal proteases and the PGRN binding partner prosaposin leads to alterations in the ratios between individual granulin peptides. Granulins B, C and E are heavily glycosylated and the glycosylation patterns can be regulated. CONCLUSION: Our results support that the levels of individual granulin peptides are differentially regulated under physiological and pathological conditions and provide novel insights into how granulin peptides function in the lysosome.

An Automated Toolbox to Predict Single Subject Atrophy in Presymptomatic Granulin Mutation Carriers.

BACKGROUND: Magnetic resonance imaging (MRI) measures may be used as outcome markers in frontotemporal dementia (FTD). OBJECTIVES: To predict MRI cortical thickness (CT) at follow-up at the single subject level, using brain MRI acquired at baseline in preclinical FTD. METHODS: 84 presymptomatic subjects carrying Granulin mutations underwent MRI scans at baseline and at follow-up (31.2±16.5 months). Multivariate nonlinear mixed-effects model was used for estimating individualized CT at follow-up based on baseline MRI data. The automated user-friendly preGRN-MRI script was coded. RESULTS: Prediction accuracy was high for each considered brain region (i.e., prefrontal region, real CT at follow-up versus predicted CT at follow-up, mean error ≤1.87%). The sample size required to detect a reduction in decline in a 1-year clinical trial was equal to 52 subjects (power = 0.80, alpha = 0.05). CONCLUSION: The preGRN-MRI tool, using baseline MRI measures, was able to predict the expected MRI atrophy at follow-up in presymptomatic subjects carrying GRN mutations with good performances. This tool could be useful in clinical trials, where deviation of CT from the predicted model may be considered an effect of the intervention itself.

Clonorchis sinensis Granulin Promotes Malignant Transformation of Hepatocyte Through EGFR-Mediated RAS/MAPK/ERK and PI3K/Akt Signaling Pathways.

The biological functions of growth factor, such as granulins, have been explored in parasites, and we elucidated that Clonorchis sinensis granulin (CsGRN) promoted the metastasis of hepatocellular carcinoma in our previous study. However, it is still unclear for the malignant transformation role of CsGRN in normal human hepatocytes. In this study, by transfecting pEGFP-C1-CsGRN eukaryotic expression plasmid, a cell line with stable overexpression of CsGRN in normal hepatocyte (LO2-GRN cells) was constructed. The effects on cell proliferation were detected by carrying out cell counting kit-8 (CCK8) assay and colony formation assay. Additionally, we conducted flow cytometry analysis to determine whether the proliferation of CsGRN was due to cell cycle arrest. Subsequently, the migration ability and the invasion ability of LO2-GRN cells were evaluated through wound-healing assay and transwell assay. Meanwhile, the levels of the markers of RAS/MAPK/ERK and PI3K/Akt signaling pathways activation in LO2-GRN cells were assessed by quantitative RT-PCR and Western blot. Our results indicated that CsGRN promoted the proliferation of LO2 cells by regulating the expression of cell-cycle-related genes. Moreover, the overexpression of CsGRN regulates malignant metastasis of liver cells by inducing the upregulation of epithelial-mesenchymal transition (EMT) marker proteins. Furthermore, both mRNA and protein expression levels of p-EGFR, RAS, p-ERK, p-AKT, p-PI3K, and p-braf have been enhanced by CsGRN. These results showed that CsGRN promoted the malignant transformation of hepatocytes by regulating epidermal growth factor receptor (EGFR)-mediated RAS/MAPK/ERK and PI3K/Akt signaling pathways, which suggested that CsGRN could serve as a novel oncoprotein during Clonorchis sinensis-associated malignant transformation of hepatocytes.

Progranulin/GP88, A Complex and Multifaceted Player of Tumor Growth by Direct Action and via the Tumor Microenvironment.

Investigation of the role of progranulin/GP88 on the proliferation and survival of a wide variety of cells has been steadily increasing. Several human diseases stem from progranulin dysregulation either through its overexpression in cancer or its absence as in the case of null mutations in some form of frontotemporal dementia. The present review focuses on the role of progranulin/GP88 in cancer development, progression, and drug resistance. Various aspects of progranulin identification, biology, and signaling pathways will be described. Information will be provided about its direct role as an autocrine growth and survival factor and its paracrine effect as a systemic factor as well as via interaction with extracellular matrix proteins and with components of the tumor microenvironment to influence drug resistance, migration, angiogenesis, inflammation, and immune modulation. This chapter will also describe studies examining progranulin/GP88 tumor tissue expression as well as circulating level as a prognostic factor for several cancers. Due to the wealth of publications in progranulin, this review does not attempt to be exhaustive but rather provide a thread to lead the readers toward more in-depth exploration of this fascinating and unique protein.

Microglial lysosome dysfunction contributes to white matter pathology and TDP-43 proteinopathy in GRN-associated FTD.

Loss-of-function mutations in the progranulin gene (GRN), which encodes progranulin (PGRN), are a major cause of frontotemporal dementia (FTD). GRN-associated FTD is characterized by TDP-43 inclusions and neuroinflammation, but how PGRN loss causes disease remains elusive. We show that Grn knockout (KO) mice have increased microgliosis in white matter and an accumulation of myelin debris in microglial lysosomes in the same regions. Accumulation of myelin debris is also observed in white matter of patients with GRN-associated FTD. In addition, our findings also suggest that PGRN insufficiency in microglia leads to impaired lysosomal-mediated clearance of myelin debris. Finally, Grn KO mice that are deficient in cathepsin D (Ctsd), a key lysosomal enzyme, have augmented myelin debris and increased neuronal TDP-43 pathology. Together, our data strongly imply that PGRN loss affects microglial activation and lysosomal function, resulting in the accumulation of myelin debris and contributing to TDP-43 pathology.

Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration.

BACKGROUND: Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases to process PGRN at a range of pH setpoints. RESULTS: In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in distinctive, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn showed increased PGRN processing to granulin F and increased AEP activity in degenerating brain regions but not in regions unaffected by disease. CONCLUSIONS: This study demonstrates that multiple lysosomal proteases may work in concert to liberate multi-granulin fragments and granulins. It also implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage and granulin production may represent therapeutic strategies for FTLD-Pgrn and other progranulin-related diseases.

Granulin: An Invasive and Survival-Determining Marker in Colorectal Cancer Patients.

BACKGROUND: Granulin is a secreted, glycosylated peptide-originated by cleavage from a precursor protein-which is involved in cell growth, tumor invasion and angiogenesis. However, the specific prognostic impact of granulin in human colorectal cancer has only been studied to a limited extent. Thus, we wanted to assess the expression of granulin in colorectal cancer patients to evaluate its potential as a prognostic biomarker. METHODS: Expressional differences of granulin in colorectal carcinoma tissue (n = 94) and corresponding healthy colon mucosa were assessed using qRT-PCR. Immunohistochemistry was performed in colorectal cancer specimens (n = 97), corresponding healthy mucosa (n = 47) and colorectal adenomas (n = 19). Subsequently, the results were correlated with histopathological and clinical patients' data. HCT-116 cells were transfected with siRNA for invasion and migration assays. RESULTS: Immunohistochemistry and qRT-PCR revealed tumoral over expression of granulin in colorectal cancer specimens compared to corresponding healthy colon mucosa and adenomas. Tumoral overexpression of granulin was associated with a significantly impaired overall survival. Moreover, downregulation of granulin by siRNA significantly diminished the invasive capacities of HCT-116 cells in vitro. CONCLUSION: Expression of granulin differs in colorectal cancer tissue, adenomas and healthy colon mucosa. Furthermore, granulin features invasive and migrative capabilities and overexpression of granulin correlates with a dismal prognosis. This reveals its potential as a prognostic biomarker and granulin could be a worthwhile molecular target for individualized anticancer therapy.

Circular RNA circ_0008274 upregulates granulin to promote the progression of hepatocellular carcinoma via sponging microRNA -140-3p.

Circular RNA (circRNA) features prominently in the progression of hepatocellular carcinoma (HCC), of which the biological function and potential mechanism of circ_0008274 in HCC are obscure. The present study aims to explore circ_ 0008274's biological functions and underlying mechanisms in HCC. The expressions of circ_0008274, miR-140-3p and Granulin (GRN) mRNA in HCC tissues and cells were investigated by quantitative real-time polymerase chain reaction. Besides, GRN protein expression was measured by Western blot. Furthermore, chi-square test was used to probe the interrelation between circ_0008274 expression and clinicopathological parameters. In addition, cell counting kit-8 (CCK-8) and EdU assays were applied to detect cell proliferation. Moreover, transwell assay was used to detect cell migration and invasion. What's more, bioinformatics prediction, dual-luciferase reporter gene assay and RNA Immunoprecipitation experiments were used to corroborate the targeting interrelations among circ_0008274, miR-140-3p and GRN. Herein we reported that circ_0008274 was highly expressed in HCC, and its high expression enhanced the proliferation, migration, and invasion of HCC cells, while depleting circ_0008274 inhibited the malignant biological behaviors of HCC cells. Mechanistically, circ_0008274 upregulates GRN expressions via adsorbing miR-140-3p to expedite the progression of HCC.