Small Leucine-rich Repeat Proteoglycan Research Articles

Abstract 4208: In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes

Abstract Preclinical models of Sonic Hedgehog (SHH)-driven tumors support the therapeutic benefit of pharmacological inhibitors, yet clinical outcomes are conflicted due to biphasic tumor responses that include complete remission or faster disease progression. In such studies, the activity of the SHH pathway in the tumor-adjacent stroma supported or restrained tumor progression. Our prior studies show that altered SHH ligand levels can lead to faster tumor growth during pharmacological inhibition suggesting a role for the SHH pathway strength in the biphasic tumor responses of a triple-negative breast cancer (TNBC) model. In this study, the role of SHH pathway strength altered by tumor-fibroblast proximity was examined on the tumor growth, invasion, and transcriptional activity using in vitro and in vivo models. Tumor spheroid models composed of TNBC cell lines and fibroblasts were used to monitor SHH signal strength as a function of the culture modality and SHH ligand levels. In vitro, SHH signal strength was altered by tumor-fibroblast proximity and ligand concentration. Mixed cultures exhibited significantly higher SHH activity and reduced invasion than adjacent cultures. This effect was reversed by pharmacological inhibition of SMO, confirming the involvement of an SHH-dependent mechanism. In a tumor xenograft model, tumor metastasis outcomes were suppressed by pharmacological inhibition of the SHH pathway, but opposite results were observed in tumor xenografts composed of co-injected with fibroblasts indicating that the biphasic tumor response can be driven by the fibroblasts. Spatial transcriptional analysis identified extracellular matrix (ECM) activities in fibroblasts as the top altered bioprocesses in both in vitro and in vivo. This finding was confirmed in cultures, where fibroblast-derived decellularized matrix stimulated tumor invasion of single TNBC spheroids at levels observed in co-cultures. Further analysis of differentially expressed genes (DEGs) identified a small leucine-rich repeat proteoglycan (SLRP) as the primary ECM target altered by variations in SHH signal strength in fibroblasts. Furthermore, we confirmed the role of SLRP cues in governing tumor invasion potency within spheroid cultures embedded in a collagen hydrogel matrix. While high levels of SLRP in the matrix inhibited spheroid invasion, low SLRP matrix promoted spheroid invasion and increased secondary invasion sites. These findings agreed with the Vimentin expression levels observed in TNBC cells and confirmed the potency of SLRP in regulating tumor invasion outcomes. In summary, our study underscores the significance of ECM remodeling activities in fibroblasts and the role of SLRPs in governing tumor invasion, with implications for understanding the impact of altering SHH signaling activity in TNBC. Citation Format: Emanuel A. Carrasquillo-Dones, Heizel M. Rosado-Galindo, Ana M. Reyes-Ramos, Wandaliz Torres-Garcia, Said Cifuentes, Jan P. Rios-Grant, Gabriela Ortiz-Soto, Natalia A. Ramos-Acevedo, Miosotis Acevedo-Esquilin, Monica Colon-Vargas, Israel Almodovar-Rivera, Camilo Mora, Michelle M. Martinez-Montemayor, Maribella Domenech. In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4208.

Low- and High-Grade Glioma-Associated Vascular Cells Differentially Regulate Tumor Growth.

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of protumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express protumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wild-type (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to antiangiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and antiangiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo cotransplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wild-type HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFβ1-GPM6A signaling.

Bone Biomarker in Feedlot Cattle from Two Different Production Systems.

The rapid growth rate in feedlot cattle is likely to promote joint abnormalities like osteochondrosis dissecans (OCD) and osteoarthritis (OA) with subsequent lameness. We have identified a small leucine-rich repeat proteoglycan (SLRP) biomarker at cleavage site 262GLGHNQIRM (BGN262) arising from the fragmentation of biglycan (BGN) in subchondral bone associated with OA. With a validated custom-made ELISA, BGN262 has been quantified in serum from cattle. The concentration of BGN262 in serum from cattle raised in the conventional and all-natural production systems increased at harvest compared to the starting period. The limitation of the study is the small sample size. However, the promising results encourage a further evaluation of BGN262 and its potential as a biomarker for subchondral bone pathology in cattle.

Structural Insights into Protein-Ligand Interactions of Small Leucine Rich Repeat Proteoglycans with a Large Number of Binding Partners: An Overview

Small leucine rich repeat proteoglycans (SLRPs) exist in the extracellular matrix. SLRPs contain tandem arrays of LRRs flanked by cysteine clusters at the both N- and C-termini. The extreme N- and/or C-termini contain low complexity sequences, glycosaminoglycan (GAG) chain and/or sulfated tyrosine residues in some members of SLRPs. The LRR solenoid structure may be divided into four parts consisting of a concave surface, an ascending surface, a convex surface, and a descending surface. SLRPs share many biological functions including collagen fibrillogenesis, signaling, innate immunity, and inflammation through the binding of various ligands. We undertake a comprehensive literature search of publications in order to make a list of ligands of SLRPs. We describe and discuss the interacting sites of SLRPs to binding partners. The protein-ligand interactions occur on not only the concave surface but also the ascending surface and the N- or C-terminal capping regions. In addition, the extreme N- and/or C-terminal regions with the GAG chains or sulfated tyrosine residues participate in ligand-interactions.

Glycomics by ion mobility tandem mass spectrometry of chondroitin sulfate disaccharide domain in biglycan.

Biglycan (BGN), a small leucine-rich repeat proteoglycan, is involved in a variety of pathological processes including malignant transformation, for which the upregulation of BGN was found related to cancer cell invasiveness. Because the functions of BGN are mediated by its chondroitin/dermatan sulfate (CS/DS) chains through the sulfates, the determination of CS/DS structure and sulfation pattern is of major importance. In this study, we have implemented an advanced glycomics method based on ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS) to characterize the CS disaccharide domains in BGN. The high separation efficiency and sensitivity of this technique allowed the discrimination of five distinct CS disaccharide motifs, of which four irregulated in their sulfation pattern. For the first time, trisulfated unsaturated and bisulfated saturated disaccharides were found in BGN, the latter species documenting the non-reducing end of the chains. The structural investigation by IMS MS/MS disclosed that in one or both of the CS/DS chains, the non-reducing end is 3-O-sulfated GlcA in a rather rare bisulfated motif having the structure 3-O-sulfated GlcA-4-O-sulfated GalNAc. Considering the role played by BGN in cancer cell spreading, the influence on this process of the newly identified sequences will be investigated in the future.

Relationship between asporin and extracellular matrix behavior: A literature review.

Asporin (ASPN), as a member of the small leucine-rich repeat proteoglycan family, is a type of protein that is found in the extracellular matrix. Collagen deposition or transformation is involved in a variety of pathological processes. ASPN is identified in cancerous tissue, pathological cardiac tissue, articular cartilage, keloid, and fibrotic lung tissue, and it has a role in the development of cancer, cardiovascular, bone and joint, keloid, and pulmonary fibrosis by interfering with collagen metabolism. This review article summarizes the data on ASPN expressions in mouse and human and highlights that overexpress of ASPN might play a role in a variety of diseases. Although our knowledge of ASPN is currently limited, these instances may help us better understand how it interacts with diseases.

TMIC-75. TUMOR ENDOTHELIAL CELL-DERIVED ASPORIN DIFFERENTIALLY REGULATES GLIOBLASTOMA GROWTH

Abstract Gliomas are the most prevalent and malignant brain tumors. A major histopathological feature that distinguishes the high-grade (IV) glioblastoma (GBM) from slow-growing low-grade (II and III) glioma is microvascular hyperproliferation. Although gliomas are highly vascularized tumors, anti-angiogenic therapies have failed to prevent recurrence and significantly improve patient survival. To determine if tumor endothelial cells (TEC) display molecular heterogeneity, we performed bulk RNA-sequencing on cultured CD31+ TEC from low-grade (II/III), and high-grade (IV, primary and recurrent) gliomas. Low- and high-grade TEC exhibited significant molecular heterogeneity, with increased enrichment of extracellular matrix and cell cycle-related gene sets in low-grade and enrichment of cytokine and immune response-related gene sets in high-grade TEC. We also found significant functional differences in proliferation capacity, and resistance to anti-angiogenic therapies between low-and high-grade TEC. Co-transplantation of low-grade TEC with tumor cells significantly inhibited GBM growth and enhanced survival, whereas high-grade TEC promoted growth of tumors in orthotopic xenografts in vivo. Differential gene expression analysis revealed several proteoglycans distinctly expressed by low-grade TEC. Of the highly enriched molecules in low-grade TEC, Asporin (ASPN), a small leucine-rich repeat proteoglycan (SLRP) significantly inhibited growth and proliferation of IDH1-wt GBM cells but did not affect IDH1-mutant tumor cells. Co-transplantation of TEC-lacking ASPN with IDH1-wt GBM partially rescued the growth-inhibitory effect of low-grade TEC on tumor growth and reduced survival. Ongoing experiments are aimed at determining the molecular mechanism underlying the growth-inhibitory effects of TEC-derived Asporin on glioma.

Expression and the Prognostic Value of Biglycan in Gastric Cancer.

Background Biglycan (BGN) is a family member of small leucine-rich repeat proteoglycans. High expression of BGN might enhance the invasion and metastasis in some types of tumors. Here, the prognostic significance of BGN was evaluated in gastric cancer. Material and Methods. Two independent Gene Expression Omnibus (GEO) gastric cancer microarray datasets (n = 64 and n = 432) were collected for this study. Kaplan-Meier analysis was applied to evaluate if BGN impacts the outcomes of gastric cancer. Protein-protein interaction (PPI) analysis was performed on gastric cancer-related genes and BGN targets, and those interactions with confidence interval (CI) ≥ 0.7 were chosen to construct a PPI network. The gene set enrichment analysis (GSEA) was used to explore BGN and cancer-related gene signatures. Gene Transcription Regulation Database (GTRD) and ALGGEN-PROMO predicted the transcription factor binding sites (TFBSs) of the BGN promoter. BGN protein level in gastric cancer tissue was determined by immunohistochemistry (IHC). Bioinformatic analysis predicted the putative TFs of BGN. Results For gastric cancer, the mRNA expression level of BGN in tumor tissue was significantly higher than that in normal tissue. Kaplan-Meier analysis showed that higher expression of BGN mRNA was significantly associated with more reduced recurrence-free survival (RFS). GSEA results suggested that BGN was significantly enriched in gene signatures related to metastasis and poor prognosis, revealing that BGN might be associated with cell proliferation, poor differentiation, and high invasiveness of gastric cancer. Meanwhile, the putative TFs, including AR, E2F1, and TCF4, were predicted by bioinformatic analysis and also significantly correlated with expression of BGN in mRNA levels. Conclusion High expression of BGN mRNA was significantly related to poor prognosis, which suggested that BGN was a potential prognostic biomarker and therapeutic target of gastric cancer.

Age-Related Alterations in the Testicular Proteome of a Non-Human Primate.

Aging of human testis and associated cellular changes is difficult to assess. Therefore, we used a translational, non-human primate model to get insights into underlying cellular and biochemical processes. Using proteomics and immunohistochemistry, we analyzed testicular tissue of young (age 2 to 3) and old (age 10 to 12) common marmosets (Callithrix jacchus). Using a mass spectrometry-based proteomics approach, we identified 63,124 peptides, which could be assigned to 5924 proteins. Among them, we found proteins specific for germ cells and somatic cells, such as Leydig and Sertoli cells. Quantitative analysis showed 31 differentially abundant proteins, of which 29 proteins were more abundant in older animals. An increased abundance of anti-proliferative proteins, among them CDKN2A, indicate reduced cell proliferation in old testes. Additionally, an increased abundance of several small leucine rich repeat proteoglycans and other extracellular matrix proteins was observed, which may be related to impaired cell migration and fibrotic events. Furthermore, an increased abundance of proteins with inhibitory roles in smooth muscle cell contraction like CNN1 indicates functional alterations in testicular peritubular cells and may mirror a reduced capacity of these cells to contract in old testes.

Sequence features, structure, ligand interaction, and diseases in small leucine rich repeat proteoglycans.

Small leucine rich repeat proteoglycans (SLRPs) are a group of active components of the extracellular matrix in all tissues. SLRPs bind to collagens and regulate collagen fibril growth and fibril organization. SLRPs also interact with various cytokines and extracellular compounds, which lead to various biological functions such cell adhesion and signaling, proliferation, and differentiation. Mutations in SLRP genes are associated with human diseases. Now crystal structures of five SLRPs are available. We describe some features of amino acid sequence and structures of SLRPs. We also review ligand interactions and then discuss the interaction surfaces. Furthermore, we map mutations associated with human diseases and discuss possible effects on structures by the mutations.

Proteomic identification and validation of novel interactions of the putative tumor suppressor PRELP with membrane proteins including IGFI-R and p75NTR

Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP’s links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.

Lumican promotes joint fibrosis through TGF-β signaling.

Joint contracture (also known as arthrofibrosis) is a fibrotic joint disorder characterized by excessive collagen production to form fibrotic scar tissue and adhesions within joint capsules. This can severely affect day-to-day activities and quality of life because of a restricted range of motion in affected joints. The precise pathogenic mechanism underlying joint contractures is not fully understood. Lumican belongs to the class II small leucine-rich repeat proteoglycan superfamily, which makes up collagen fibrils in the extracellular matrix. Lumican is ubiquitously expressed in the skin, liver, heart, uterus and articular cartilage and has reported roles in cell migration, proliferation, angiogenesis and Toll-like receptor 4 signaling. Previous research has suggested that lumican is involved in the pathogenesis of several fibrotic diseases. Because joint contracture resembles a fibrotic disease, we aimed to investigate the role of lumican in the development of joint contracture invitro. Here, we showed that protein levels were up-regulated in the fibrotic joint capsule versus control. We observed that lumican significantly enhanced the proliferation, migration and fibroblast-myofibroblast transition of synovial fibroblasts. Moreover, lumican led to increased transcription of alpha-smooth muscle actin, matrix metallopeptidase 9, Collagen I, plasminogen activator inhibitor 1 and transforming growth factor-β invitro. Lumican treatment promoted collagen lattice contraction in a dose-dependent manner as early as 24h after treatment. Thus, our studies reveal that lumican could promote fibroblast-myofibroblast transition and joint contracture.

Adding exogenous biglycan or decorin improves tendon formation for equine peritenon and tendon proper cells in vitro

BackgroundTendon injuries amount to one of the leading causes of career-ending injuries in horses due to the inability for tendon to completely repair and the high reinjury potential. As a result, novel therapeutics are necessary to improve repair with the goal of decreasing leg lameness and potential reinjury. Small leucine-rich repeat proteoglycans (SLRPs), a class of regulatory molecules responsible for collagen organization and maturation, may be one such therapeutic to improve tendon repair. Before SLRP supplementation can occur in vivo, proper evaluation of the effect of these molecules in vitro needs to be assessed. The objective of this study was to evaluate the effectiveness of purified bovine biglycan or decorin on tendon proper and peritenon cell populations in three-dimensional tendon constructs.MethodsEquine tendon proper or peritenon cell seeded fibrin three-dimensional constructs were supplemented with biglycan or decorin at two concentrations (5 nM or 25 nM). The functionality and ultrastructural morphology of the constructs were assessed using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression by real time – quantitative polymerase chain reaction (RT-qPCR).ResultsSLRP supplementation affected both tendon proper and peritenon cells-seeded constructs. With additional SLRPs, material and tensile properties of constructs strengthened, though ultrastructural analyses indicated production of similar-sized or smaller fibrils. Overall expression of tendon markers was bolstered more in peritenon cells supplemented with either SLRP, while supplementation of SLRPs to TP cell-derived constructs demonstrated fewer changes in tendon and extracellular matrix markers. Moreover, relative to non-supplemented tendon proper cell-seeded constructs, SLRP supplementation of the peritenon cells showed increases in mechanical strength, material properties, and collagen content.ConclusionsThe SLRP-supplemented peritenon cells produced constructs with greater mechanical and material properties than tendon proper seeded constructs, as well as increased expression of matrix assembly molecules. These findings provide evidence that SLRPs should be further investigated for their potential to improve tendon formation in engineered grafts or post-injury.

BiAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

3046High levels of MMP-cleaved mimecan is associated to carotid plaque stability and less future cardiovascular events

Abstract Introduction The clinical consequences of atherosclerosis, myocardial infarction and stroke are the most common causes of death globally. Mimecan, a small leucine rich-repeat proteoglycan (SLRP), is cleaved by matrix metalloproteinases (MMPs) and known to be involved in collagen fibrillogenesis and angiogenesis. Circulating levels of MMP-cleaved mimecan (cMIM) has previously been identified as a marker of extracellular matrix remodelling in ApoE−/− knockout mice. The role of mimecan and its degradation in human atherosclerotic plaques has not been explored. Purpose We explored whether full-length mimecan and cleaved mimecan (cMIM) are associated to plaque composition and evaluated if they can predict future cardiovascular events. Methods Two hundred and eighteen human atherosclerotic plaques were stained for mimecan using immunohistochemistry. cMIM was measured in 202 plaque tissue homogenates using a competitive ELISA assay. Histological components (α-actin, CD68 and glycophorin A) were assessed using immunohistochemistry, neutral lipids were measured using Oil Red O and visible areas of calcium deposits were quantified. Matrix metalloproteinases (MMP-1, -2, -3, -9, -10 and -12), tissue inhibitors of matrix metalloproteinases (TIMP-1 and -2) were analysed in plaque tissue homogenates using ELISA assays and a proximity extension assay. ECM components (glycosaminoglycans, collagen and elastin) were detected with colorimetric assays and the TGF-β1, β2 and β3 were measured by a multiplex assay. Cardiovascular events were registered using national registers, patient records and telephone calls during a follow-up period of 59 months IQR (34–73). Results Mimecan was expressed in human atherosclerotic plaques. The expression correlated positively with neutral lipids and intraplaque hemorrhage and inversely with α-actin. In contrast cMIM correlated with α-actin and inversely with neutral lipids. cMIM correlated also with stabilizing extracellular matrix proteins elastin, collagen as well as TGF-β1, β2 and β3. Mimecan correlated to MMP-9 and cMIM correlated to MMP-2 and TIMP-2. Patient with high levels of cMIM had a lower risk of future cardiovascular events which remained significant after adjusting for risk factors (age, gender, diabetes and symptoms) in a multivariate Cox regression Conclusion(s) Mimecan was associated with vulnerable plaque features, whereas cMIM was related to stable plaque features. Low levels of cMIM predicted future cardiovascular events, independently of known risk factors. Taken together this suggests a possible role for mimecan and its cleavage in atherosclerosis that needs to be further explored. Acknowledgement/Funding Danish Research Foundation, Swedish Research Council, Swedish Heart and Lung Foundation, Skåne University Hospital and more.

Significance of Tsukushi in lung cancer

ObjectivesTsukushi (TSK), a member of the small leucine-rich repeat proteoglycan (SLRP) family, plays multifunctional roles by interacting with signaling molecules during development. However, the role of TSK in cancer remains unknown. The aim of the present study was to investigate the biological significance of TSK in lung cancer. Materials and methodsImmunohistochemistry of lung cancer tissues and reverse transcription polymerase chain reaction (PCR) of lung cancer cell lines were carried out to detect TSK. Then, RNA sequence analysis, Gene Ontology analysis, quantitative real-time PCR, western blotting, cell counting assay, invasion assays, and xenograft studies were done in a human lung adenocarcinoma cell line, H1975 with modification of TSK expression levels, in order to investigate its biological roles, in particular epithelial–mesenchymal transition (EMT) and proliferation. ResultsTSK was found to be highly expressed in lung cancer tissues and cell lines. Modification of TSK expression levels in H1975 resulted in changes in molecules related to EMT, including cadherin-1, snail family transcriptional repressor 1, snail family transcriptional repressor 2, and vimentin. The results of cell counting and xenograft assays showed that TSK promotes cell proliferation. ConclusionsIn lung cancer cells, TSK is expressed more highly than the other SLRPs family members, and regulates the EMT and proliferation. Thus, TSK may be a key coordinator of multiple pathways and an important structural element in the lung cancer microenvironment.

Leucine Rich Repeat Proteins: Sequences, Mutations, Structures and Diseases

Mutations in the genes encoding Leucine Rich Repeat (LRR) containing proteins are associated with over sixty human diseases; these include high myopia, mitochondrial encephalomyopathy, and Crohn's disease. These mutations occur frequently within the LRR domains and within the regions that shield the hydrophobic core of the LRR domain. The amino acid sequences of fifty-five LRR proteins have been published. They include Nod-Like Receptors (NLRs) such as NLRP1, NLRP3, NLRP14, and Nod-2, Small Leucine Rich Repeat Proteoglycans (SLRPs) such as keratocan, lumican, fibromodulin, PRELP, biglycan, and nyctalopin, and F-box/LRR-repeat proteins such as FBXL2, FBXL4, and FBXL12. For example, 363 missense mutations have been identified. Replacement of arginine, proline, or cysteine by another amino acid, or the reverse, is frequently observed. The diverse effects of the mutations are discussed based on the known structures of LRR proteins. These mutations influence protein folding, aggregation, oligomerization, stability, protein-ligand interactions, disulfide bond formation, and glycosylation. Most of the mutations cause loss of function and a few, gain of function.

Asporin promotes cell proliferation via interacting with PSMD2 in gastric cancer.

Asporin (ASPN), a member of small leucine-rich repeat proteoglycan (SLRP) family of proteins, serves important roles in diverse biological responses and disease conditions. We tested the hypothesis that ASPN regulated proliferation of gastric cancer (GC) cells and identified its down-stream regulators. ASPN promoted the proliferation of GC cells. We identified the effector of this effect as proteasome 26S subunit non-ATPase 2 (PSMD2) which is known to regulate proliferation through suppression of DUSP7, WIP1 and PTEN and then inducing the phosphorylation of ERK, P38 and AKT. PSMD2 co-immunoprecipitated with ASPN from GC cell lysates and co-localized with PSMD2 inside GC cells. Moreover, knockdown of ASPN significantly increased the expression of DUSP7, WIP1 and PTEN and led to a repression in the phosphorylation of ERK, P38 and AKT. These changes were counteracted by knockdown of PSMD2. In conclusion, ASPN promotes cell proliferation by interacting with PSMD2, and down-regulation of its effectors and serves as a potential therapeutic target in GC.

AFM Characterization of the Internal Mammary Artery as a Novel Target for Arterial Stiffening.

Using the atomic force microscopy- (AFM-) PeakForce quantitative nanomechanical mapping (QNM) technique, we have previously shown that the adventitia of the human internal mammary artery (IMA), tested under dehydrated conditions, is altered in patients with a high degree of arterial stiffening. In this study, we explored the nanoscale elastic modulus of the tunica media of the IMA in hydrated and dehydrated conditions from the patients with low and high arterial stiffening, as assessed in vivo by carotid-femoral pulse wave velocity (PWV). In both hydrated and dehydrated conditions, the medial layer was significantly stiffer in the high PWV group. The elastic modulus of the hydrated and dehydrated tunica media was significantly correlated with PWV. In the hydrated condition, the expression activity of certain small leucine-rich repeat proteoglycans (SLRPs), which are associated with arterial stiffening, were found to be negatively correlated to the medial elastic modulus. We also compared the data with our previous work on the IMA adventitia. We found that the hydrated media and dehydrated adventitia are both suitable for reflecting the development of arterial stiffening and SLRP expression. This comprehensive study of the nanomechanical properties integrated with the proteomic analysis in the IMAs demonstrates the possibility of linking structural properties and function in small biological samples with novel AFM methods. The IMA is a suitable target for predicting arterial stiffening.

Serum decorin is a potential prognostic biomarker in patients with acute exacerbation of idiopathic pulmonary fibrosis.

Decorin is a small leucine-rich repeat proteoglycan that plays a critical role in collagen fibrillogenesis, and regulates inflammation, wound healing and angiogenesis. In idiopathic pulmonary fibrosis (IPF), decorin is expressed in fibrotic lesions; furthermore, intratracheal gene transfer of decorin has been demonstrated to inhibit bleomycin-induced pulmonary fibrosis. Although these results suggest the critical role of decorin in pulmonary fibrosis, the role of decorin in the acute exacerbation of idiopathic interstitial pneumonia (AE-IIP) has not been clarified in detail. Thus, the goal of this study was to determine the role of decorin in AE-IIP. We retrospectively analyzed AE-IIP patients who had been admitted to our hospital. First, serum decorin levels were compared among patients with AE-IIP, patients with stable idiopathic interstitial pneumonia (SD-IIP), and healthy subjects. Next, the relationship between serum decorin levels and clinical parameters was analyzed in AE-IIP patients. Finally, the association between serum decorin levels and prognosis was evaluated in AE-IIP patients. IIP was divided into IPF and non-IPF, according to the published guidelines. The serum decorin levels of AE-IIP patients were significantly lower than those of both healthy subjects and SD-IIP patients. Serum decorin levels were not related with the clinical parameters and prognosis, when all IIP patients were analyzed. In IPF patients, serum decorin levels had a significant correlation with oxygenation, and IPF patients with low serum decorin levels had a significantly higher survival rate than those with high serum decorin levels. Serum decorin levels are a potential prognostic biomarker in AE-IPF.