Reversion-inducing Cysteine-rich Protein Research Articles

Chronic intermittent hypoxia facilitates the development of angiotensin II-induced abdominal aortic aneurysm in male mice.

Obstructive sleep apnea (OSA), characterized by episodes of intermittent hypoxia (IH), is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, whether IH serves as an independent risk factor for AAA development remains to be investigated. Here, we determined the effects of chronic (6 mo) IH on angiotensin (Ang II)-induced AAA development in C57BL/6J male mice and investigated the underlying mechanisms of IH in cultured vascular smooth muscle cells (SMCs). IH increased the susceptibility of mice to develop AAA in response to Ang II infusion by facilitating the augmentation of the abdominal aorta's diameter as assessed by transabdominal ultrasound imaging. Importantly, IH with Ang II augmented aortic elastin degradation and the expression of matrix metalloproteinases (MMPs), mainly MMP8, MMP12, and a disintegrin and metalloproteinase-17 (ADAM17) as measured by histology and immunohistochemistry. Mechanistically, IH increased the activities of MMP2, MMP8, MMP9, MMP12, and ADAM17, while reducing the expression of the MMP regulator reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in cultured SMCs. Aortic samples from human AAA were associated with decreased RECK and increased expression of ADAM17 and MMPs. These data suggest that IH facilitates AAA development when additional stressors are superimposed and that this occurs in association with an increased presence of aortic MMPs and ADAM17, potentially due to IH-induced modulation of RECK expression. These findings support a plausible synergistic link between OSA and AAA and provide a better understanding of the molecular mechanisms underlying the pathogenesis of AAA.NEW & NOTEWORTHY IH facilitates Ang II-induced abdominal aortic diameter expansion and AAA development in C57BL/6J male mice. IH upregulates the expression of specific MMPs such as MMP8, MMP12, and ADAM17. IH directly suppresses RECK expression and increases MMPs activity in SMCs. Human AAA tissues exhibit a downregulation of RECK and an upregulation of ADAM17 and MMPs.

SIAH1 facilitates the migration and invasion of gastric cancer cells through promoting the ubiquitination and degradation of RECK

Siah E3 ubiquitin protein ligase 1 (SIAH1) has been reported to participate in the development of several human cancers, including gastric cancer. However, the effect and mechanism of SIAH1 on the migration and invasion of gastric cancer cells need be further explored. Here, we first analyzed the clinical value of SIAH1 in gastric cancer, and found that SIAH1 was up-regulated in gastric cancer and associated with a poor prognosis. In addition, silencing of SIAH1 significantly inhibited the migration and invasion of gastric cancer cells through inhibiting the expression of matrix metalloproteinase-9 (MMP9), while overexpression of SIAH1 had the opposite effect. Molecularly, we provided the evidence that reversion-inducing cysteine-rich protein with Kazal motifs (RECK) was a potential substrate of SIAH1. We determined that SIAH1 could destabilize RECK through promoting its ubiquitination and degradation via proteasome pathway. We also found RECK was involved in SIAH1-regulated gastric cancer cell migration and invasion. In conclusion, SIAH1 is up-regulated in gastric cancer, which promotes the migration and invasion of gastric cancer cells through regulating RECK-MMP9 pathway.

Multi-Functional Role of RECK in Metabolic Dysfunction-Associated Steatotic Liver Disease Development: A Mass Spectrometry-Proteomic Approach

Metabolic dysfunction-associated steatotic liver disease (MASLD, previously referred to as nonalcoholic fatty liver disease) is characterized by increased fat deposition and inflammation in the liver as well as dysregulation of cellular processes that affect the extracellular matrix (ECM). We previously observed that the expression of Reversion Inducing Cysteine Rich Protein with Kazal motifs (RECK), a key ECM regulatory protein, negatively correlates with MASLD-associated fibrosis and inflammation (metabolic dysfunction-associated steatohepatitis, MASH). However, mechanisms governing this relationship remain unclear. Therefore, we hypothesized that overexpression of RECK is hepatoprotective in the setting of MASH by altering several ECM-linked pathways, as analyzed by proteomics. Accordingly, we generated hepatocyte-specific Reck-overexpressing transgenic mice. Male hepatocyte-specific Reck-overexpressing transgenic mice and littermate controls were fed a Western diet high in fat, carbohydrate, and cholesterol for 24 weeks (n=6/group). Whole liver tissue was submitted for mass spectrometry-proteomic analysis. We identified 370 differentially expressed proteins out of a total of 4693 proteins using a q-value cutoff of ≤0.10; of which 90 proteins were significantly upregulated while 280 significantly downregulated. This included an upregulation of Cyp2b (adjusted p≤0.01) and downregulation of Taok1, C/EBPβ, and Vitronectin (all adjusted p≤0.01), all of which have been shown to be important in liver health and metabolism in the context of MASH. Predictive pathway analysis using Ingenuity Pathway Analysis software (Qiagen) identified several significantly altered pathways, including those involved in ECM regulator pathways (integrin signaling, z-score=6.0; mTOR signaling, z-score=5.3; leukocyte extravasation signaling, z-score=5.1), cell-signaling (chemokine signaling, z-score=5.4; CXCR4 signaling chemokine signaling, z-score=5.0), and inflammatory responses (leukocyte extravasation signaling, z-score=5.1; CXCR4 signaling, z-score=5.0). Interestingly, pathway analysis based on significantly altered proteins predicted the downregulation of critical components of hepatic inflammation and fibrosis, including TGF-β, IL-1β, and TNFα. These novel results reveal the regulatory role of RECK in MASH progression, specifically governing the components of ECM remodeling and inflammation. Our goal is to develop RECK inducers and identify additional therapeutic targets in MASH development and progression. This work was funded by the NIH award R01DK130243. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Over the past three decades a diverse collection of small protein domains have been used as scaffolds to generate general-purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK), and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and Next Generation Sequencing (NGS), we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low uM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were insertion into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general-purpose protein-binding reagents.

Matrix Metalloproteinases in the Periodontium-Vital in Tissue Turnover and Unfortunate in Periodontitis.

The extracellular matrix (ECM) is a complex non-cellular three-dimensional macromolecular network present within all tissues and organs, forming the foundation on which cells sit, and composed of proteins (such as collagen), glycosaminoglycans, proteoglycans, minerals, and water. The ECM provides a fundamental framework for the cellular constituents of tissue and biochemical support to surrounding cells. The ECM is a highly dynamic structure that is constantly being remodeled. Matrix metalloproteinases (MMPs) are among the most important proteolytic enzymes of the ECM and are capable of degrading all ECM molecules. MMPs play a relevant role in physiological as well as pathological processes; MMPs participate in embryogenesis, morphogenesis, wound healing, and tissue remodeling, and therefore, their impaired activity may result in several problems. MMP activity is also associated with chronic inflammation, tissue breakdown, fibrosis, and cancer invasion and metastasis. The periodontium is a unique anatomical site, composed of a variety of connective tissues, created by the ECM. During periodontitis, a chronic inflammation affecting the periodontium, increased presence and activity of MMPs is observed, resulting in irreversible losses of periodontal tissues. MMP expression and activity may be controlled in various ways, one of which is the inhibition of their activity by an endogenous group of tissue inhibitors of metalloproteinases (TIMPs), as well as reversion-inducing cysteine-rich protein with Kazal motifs (RECK).

Association between the Reduced Expression of RECK and Neutrophilic Inflammation in Chronic Obstructive Pulmonary Disease

Introduction: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a recently discovered inhibitor of matrix metalloproteinase (MMP). There is a large number of chronic obstructive pulmonary disease (COPD) patients worldwide; however, the role of RECK on COPD has not been studied. This study explored the expression of RECK in COPD patients and its effect on neutrophil function to provide a new scientific basis for the prevention and treatment of COPD. Method: Fifty patients with acute exacerbation of COPD and fifty healthy controls were enrolled in the study. RECK was detected in lung tissue, sputum, and plasma of subjects as well as in BEAS-2B cells stimulated with cigarette smoke extract (CSE) by immunohistochemistry, ELISA, and qRT-PCR. Meanwhile, lung function (FEV1%pred) and inflammatory cytokines (IL-6 and IL-8) were examined, and correlation analysis was performed with RECK expression. The effect of RECK on proliferation, apoptosis, migration, and inflammatory cytokines and its potential mechanism was further quantified by neutrophil stimulated with recombinant human RECK protein (rhRECK) combined with CSE using CCK8, flow cytometry, Transwell assay, qRT-PCR, ELISA, and Western analysis. Results: RECK was mainly expressed on airway epithelial cells in normal lung tissue and was significantly diminished in COPD patients. The levels of RECK in sputum and plasma were also significantly decreased in COPD patients. Pearson correlation analysis showed that RECK level in plasma was positively correlated with FEV1%pred (r = 0.458, p < 0.001) and negatively correlated with IL-6 and IL-8 (r = −0.386, −0.437; p = 0.006, 0.002) in COPD patients. The expression of RECK was decreased in BEAS-2B stimulated with CSE. The migration, inflammation, and MMP-9 expression of neutrophils were promoted by CSE, while inhibited by rhRECK. Conclusion: RECK is low expressed in COPD patients and negatively correlated with inflammation. It may inhibit the inflammation and migration of neutrophils by downregulating MMP-9.

Hepatocellular RECK as a Critical Regulator of Metabolic Dysfunction-associated Steatohepatitis Development

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is an extracellular matrix regulator with anti-fibrotic effects. However, its expression and role in metabolic dysfunction-associated steatohepatitis (MASH) and hepatic fibrosis are poorly understood. We generated a novel transgenic mouse model with RECK overexpression specifically in hepatocytes to investigate its role in Western diet (WD)-induced liver disease. Proteomic analysis and invitro studies were performed to mechanistically link RECK to hepatic inflammation and fibrosis. Our results show that RECK expression is significantly decreased in liver biopsies from human patients diagnosed with MASH and correlated negatively with severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and fibrosis. Similarly, RECK expression is downregulated in WD-induced MASH in wild-type mice. Hepatocyte-specific RECK overexpression significantly reduced hepatic pathology in WD-induced liver injury. Proteomic analysis highlighted changes in extracellular matrix and cell-signaling proteins. Invitro mechanistic studies linked RECK induction to reduced ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and ADAM17 activity, amphiregulin release, epidermal growth factor receptor activation, and stellate cell activation. Our invivo and mechanistic invitro studies reveal that RECK is a novel upstream regulator of inflammation and fibrosis in the diseased liver, its induction is hepatoprotective, and thus highlights its potential as a novel therapeutic in MASH.

Recent Updates on the Therapeutic Prospects of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Liver Injuries.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored glycoprotein, negatively regulates various membrane proteins involved in the tissue governing extracellular matrix (ECM) remodeling such as metalloproteases (MMPs) and the sheddases ADAM10 and ADAM17. The significance of the present review is to summarize the current understanding of the pathophysiological role of RECK, a newly discovered signaling pathway associated with different liver injuries. Specifically, this review analyzes published data on the downregulation of RECK expression in hepatic ischemia/reperfusion (I/R) injury, liver-related cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as in the progression of nonalcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). In addition, this review discusses the regulation of RECK by inducers, such as FXR agonists. The RECK protein has also been suggested as a potential diagnostic and prognostic marker for liver injury or as a biomarker with predictive value for drug treatment efficacy.

Abstract 13109: Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration

Empagliflozin, an FDA-approved anti-hyperglycemic agent, is a potent and highly selective inhibitor of sodium glucose co-transporter-2 (SGLT-2) in renal proximal tubule epithelial cells. It blocks glucose reabsorption and promotes glucose excretion via urine. Intriguingly, several clinical trials have reported the pleiotropic beneficial effects of SGLT-2 inhibitors in cardiovascular diseases, including antioxidant and anti-inflammatory effects. Here we investigated whether empagliflozin reverses oxidatively modified LDL (OxLDL)-induced matrix metalloproteinase activation and smooth muscle cell (SMC) migration and proliferation. Quiesced human aortic SMCs (Lonza) were treated with empagliflozin (1 uM) followed by human medium OxLDL (40 ug/mL; Kalen Biomedical). OxLDL stimulated SMC migration (1.8-fold, P&lt;0.05, n=4) and proliferation (1.5-fold, P&lt;0.05, n=4), and empagliflozin attenuated both by 67% (P&lt;0.05, n=4) and 61% (P&lt;0.05, n=4), respectively. Increased migration and proliferation by OxLDL were accompanied by elevated MMP-2 (3.2-fold, P&lt;0.05, n=5) and 9 (3.1-fold, P&lt;0.05, n=5) activities, which were also suppressed by empagliflozin by 65% and 67%, respectively (both P&lt;0.05, n=5). RECK (reversion inducing cysteine rich protein with Kazal motifs) is a membrane anchored MMP inhibitor, and we found that OxLDL dose-dependently downregulated RECK in SMCs (by 60%, P&lt;0.05, n=3) via NF-kB-dependent miR-30b induction. Intriguingly, empagliflozin restored the OxLDL downregulation of RECK to 88% of the control (n=3), as it inhibited miR-30b induction (by 49%, P&lt;0.05, n=5). Consistent with RECK-mediated effects of empagliflozin, adenoviral overexpression of RECK inhibited OxLDL-induced SMC migration (by 67%, P&lt;0.05, n=5) and proliferation (by 50%, P&lt;0.05, n=5). Together, these results suggest that the vasculoprotective effects of empagliflozin are at least in part mediated by restoring RECK, thereby attenuating SMC migration and proliferation under elevated oxidative stress condition. Our results also suggest therapeutic potential of empagliflozin or RECK overexpression in vascular proliferative diseases.

Identification of RECK as a protective prognostic indicator and a tumor suppressor through regulation of the ERK/MAPK signaling pathway in gastric cancer

BackgroundGastric cancer (GC) ranks as the fifth most common cancer worldwide and is characterized by its significant heterogeneity and unfavorable prognosis. Thus, identifying efficient prognostic factors and understanding the underlying molecular mechanisms in GC are essential for improving patient outcomes. In this study, we aimed to investigate the role of RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the prognostic significance and molecular mechanisms of its biological function in GC.MethodsMultiple bioinformatics strategies were performed to detect the potential functions and prognostic efficiency of RECK in GC. Rescue experiments revealed that the molecular mechanism by which RECK in inhibited tumor proliferation, migration, and invasion was mediated by ERK/MAPK signaling in AGS and HGC-27 cells. Using integrated bioinformatics analysis and western blot assay, we investigated the potential interaction between CALD1 and RECK.ResultsOur findings revealed significantly decreased RECK expression in GC samples compared to normal samples and RECK was identified as a promising predictor for the prognosis of GC patients. Moreover, upregulation of RECK demonstrated a distinctly positive association with a high-immunity and low-metastasis microenvironment in GC. Mechanistically, the antitumour effects of RECK on hampering tumor cell growth, migration, and invasion were mediated by the ERK/MAPK signaling pathway. In addition, we also illustrated that RECK inhibited the phosphorylation of CALD1 mediated by decreased phosphorylation of ERK.ConclusionsRECK is a promising prognostic biomarker and may shape a high-tumor-immunity and low-metastasis microenvironment in patients with GC. Moreover, RECK exerted its tumor-suppressive effects by the inactivation of ERK/MAPK signaling in GC cells.

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

The BAP31/miR-181a-5p/RECK axis promotes angiogenesis in colorectal cancer via fibroblast activation.

B-cell receptor-associated protein 31 (BAP31) has been recognized as a tumor-associated protein and has largely been shown to promote metastasis in a variety of cancers. Cancer metastasis arises through multistep pathways, and the induction of angiogenesis is shown to be a rate-limiting step in the process of tumor metastasis. This study explored the effect of BAP31 on colorectal cancer (CRC) angiogenesis by regulating the tumor microenvironment. First, exosomes from BAP31-regulated CRCs affected the transition of normal fibroblasts to proangiogenic cancer-associated fibroblasts (CAFs) in vivo and in vitro. Next, microRNA sequencing was performed to analyze the microRNA expression profile of exosomes secreted from BAP31- overexpressing CRCs. The results indicated that the expression of BAP31 in CRCs significantly altered the levels of exosomal microRNAs, such as miR-181a- 5p. Meanwhile, an in vitro tube formation assay showed that fibroblasts with high levels of miR-181a-5p significantly promoted endothelial cell angiogenesis. Critically, we first identified that miR-181a-5p directly targeted the 3'-untranslated region (3'UTR) of reversion-inducing cysteine-rich protein with kazal motifs (RECK) using the dual-luciferase activity assay, which drove fibroblast transformation into proangiogenic CAFs by upregulating matrix metalloproteinase-9 (MMP-9) and phosphorylation of mothers against decapentaplegic homolog 2/Mothers against decapentaplegic homolog 3 (Smad2/3). Exosomes from BAP31-overexpressing/BAP31-knockdown CRCs are found to manipulate the transition of fibroblasts into proangiogenic CAFs by the miR-181a-5p/RECK axis.

Abstract B017: Analysis of Wnt signaling and metastasis related protein in invasive ductal carcinoma

Abstract Ductal carcinoma in situ (DCIS) characterizes about 20% of all breast cancers. Breast cancer is the most common cancer among women, the number one cause of cancer mortality, and one of the leading causes of morbidity and mortality for women worldwide. Wnt signaling have been implicated in more than 45% of breast cancer patients and has been shown to decrease survival in breast cancer patients. One of the major challenges of Breast cancer treatment is that it is a heterogeneous disease comprised of four main sub types. Therefore, there is a need to investigate possible signaling pathways that may be specific for each subtype of breast cancer, so as to establish a targeted therapy and management protocol for breast cancer patients This study analyzed the expression of Wnt signaling and metastasis related protein, reversion-inducing cysteine-rich protein with kazal motif (RECK) in molecular subtypes of breast cancer samples of DCIS origin. Formalin Fixed Paraffin Embedded (FFPE) breast tumor samples were used for this study. The breast cancer tissues were divided into estrogen receptor/progesterone receptor (ER/PR) positive, ER positive, human epidermal growth factor receptor 2 (HER-2) positive, Triple negative and Triple positive based on immunohistochemical reactivity for breast cancer subtype specific antibody. These tissues were further processed for expression of Wnt signaling and metastatic related protein. Result from this study showed that Beta-catenin was highly expressed in estrogen receptor and progesterone receptor subtypes of breast cancer whereas adenomatous polyposis coli was positive for estrogen receptor and negative for the other subtypes. Furthermore, the triple negative breast cancer may metastasize earlier than other sub types of breast cancer because it has low expression of RECK when compared with other breast cancer sub-types. In conclusion, subtypes of breast cancer have different expression of Wnt signaling and metastatic related protein expression and as such Wnt signaling may correlate with metastasis in breast cancer. Citation Format: Anuoluwapo A. Adeshina, Babajide O. Ajayi. Analysis of Wnt signaling and metastasis related protein in invasive ductal carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B017.

Mycoplasma pneumoniae downregulates RECK to promote matrix metalloproteinase-9 secretion by bronchial epithelial cells

ABSTRACT Airway epithelial cells function as both a physical barrier against harmful substances and pathogenic microorganisms and as an important participant in the innate immune system. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in modulating inflammatory responses during respiratory infections. However, the signalling cascade that induces MMP-9 secretion from epithelial cells infected with Mycoplasma pneumoniae remains poorly understood. In this study, we investigated the mechanism of MMP-9 secretion in airway epithelial cells infected with M. pneumoniae. Our data clearly showed that M. pneumoniae induced the secretion of MMP-9 from bronchial epithelial cells and upregulated its enzymatic activity in a time- and dose-dependent manner. Using specific inhibitors and chromatin co-precipitation experiments, we confirmed that the expression of MMP-9 is reliant on the activation of the Toll-like receptor 2 (TLR2) and TLR6-dependent mitogen-activated protein kinase/nuclear factor- κB/activator protein-1 (MAPK/NF-κB/AP-1) pathways. Additionally, epigenetic modifications such as histone acetylation and the nuclear transcription factor Sp1 also regulate MMP-9 expression. M. pneumoniae infection also decreased the expression of the tumour suppressor reversion-inducing cysteine-rich protein with Kazal motifs (RECK) by inducing Sp1 phosphorylation. Overexpression of RECK significantly impaired the M. pneumoniae-triggered increase in MMP-9 enzymatic activity, although the level of MMP-9 protein remained constant. The study demonstrated that M. pneumoniae-triggered MMP-9 expression is modulated by TLR2 and 6, the MAPK/NF-κB/AP-1 signalling cascade, and histone acetylation, and M. pneumoniae downregulated the expression of RECK, thereby increasing MMP-9 activity to modulate the inflammatory response, which could play a role in airway remodelling.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

A Mechanistic Role for RECK in the Regulation of Hepatocellular Inflammation

RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane‐anchored glycoprotein, modifies the extracellular matrix involved in various cancers, including hepatocellular carcinoma (HCC). Its role in regulating inflammatory and fibrogenic processes has also been postulated. Both inflammation and fibrosis contribute to progression of nonalcoholic steatohepatitis (NASH) to HCC. Here, we tested the hypothesis that inducing or sustaining RECK expression will inhibit proinflammatory amphiregulin and epidermal growth factor receptor (EGFR) signaling, progression of NASH and the development of HCC using an in vitro cell culture model employing isolated adult mouse primary hepatocytes. Ectopic RECK overexpression (gain‐of‐function) was achieved by the adenoviral (Adv) transduction of murine RECK cDNA. Adv.GFP served as a control. RECK expression was silenced (loss‐of‐function) by transducing RECK‐specific siRNA using lipofectamine. Nonspecific siRNA served as a control. Hepatocytes were exposed to TNFα in the presence or absence of inhibitors against ADAM 10 (A Disintegrin and Metalloproteinase Domain‐Containing Protein 10) and/or ADAM17 – two well‐established pro‐inflammatory sheddases. Results show that RECK overexpression inhibited TNFα‐induced ADAM10/17 activity, and amphiregulin (an EGFR ligand) and EGFR expression. In the presence of ADAM10/17 inhibitors, no further reduction was observed in amphiregulin and EGFR activity, suggesting a maximal inhibitory effect of RECK on EGFR activity. In contrast, silencing RECK significantly increased amphiregulin secretion, and this effect was reversed by inhibition of ADAM10/17, indicating RECK signals via regulation of these sheddases. Thus, RECK not only regulates sheddase activity of ADAM10 and 17, but also downstream amphiregulin and EGFR signaling under pro‐inflammatory stimuli. Because increased and sustained EGFR activity contributes to progression of NASH to HCC in preclinical models, our results indicate that inducing RECK has the potential to inhibit hepatocellular inflammation in the setting of NASH and HCC.

Salvianolic acid B targets mortalin and inhibits the migration and invasion of hepatocellular carcinoma via the RECK/STAT3 pathway

BackgroundTumor migration and invasion is a complex and diverse process that involves the epithelial–mesenchymal transition (EMT) of tumor cells and degradation of the extracellular matrix by matrix metalloproteases (MMPs). Mortalin is an important oncogene. It has been reported to play an important role in tumor migration and invasion through various signaling pathways, but the underlying mechanism is not fully understood.MethodsHere, we investigated the role of mortalin in the migration of the hepatocellular carcinoma (HCC) cell lines HepG2 and HCCLM3.ResultsThe overexpression of mortalin in HepG2 cells decreased the protein level of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and activated the phosphorylation and acetylation of STAT3, thereby up-regulating matrix metalloproteinase 9 (MMP9) and promoting cell migration and invasion. In contrast, in HCCLM3 cells, mortalin knockdown increased the expression of RECK, inhibited the STAT3 pathway and the activity of MMP9, and inhibited cell migration and invasion. Furthermore, we found that salvianolic acid B, a caffeic acid phenethyl ester analog, specifically bound to mortalin and increased the degradation of mortalin proteasomes through ubiquitination, thereby up-regulating RECK, inhibiting STAT3, and finally inhibiting the migration and invasion of HCC cells.ConclusionOur work suggested that mortalin is a potential therapeutic target for hepatocellular carcinoma.

Long Non-Coding RNA MEG8 Suppresses Hypoxia-Induced Excessive Proliferation, Migration and Inflammation of Vascular Smooth Muscle Cells by Regulation of the miR-195-5p/RECK Axis

It has been recognized that rebalancing the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) helps relieve vascular injury. Presently, we aim to investigate whether long non-coding RNA (lncRNA) maternally expressed 8 (MEG8) plays a role in affecting the excessive proliferation and migration of VSMCs following hypoxia stimulation. A percutaneous transluminal angioplasty balloon dilatation catheter was adopted to establish vascular intimal injury, the levels of MEG8 and miR-195-5p in the carotid artery were tested by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Hypoxia was used to stimulate VSMCs, then the cell counting kit-8 (CCK-8) assay, Transnwell assay, and wound healing assay were conducted to evaluate the proliferation, and migration of VSMCs. The protein levels of RECK (reversion inducing cysteine rich protein with kazal motifs), MMP (matrix metalloproteinase) 3/9/13, COX2 (cytochrome c oxidase subunit II), macrophage inflammatory protein (MIP)-1beta, VCAM-1 (vascular cell adhesion molecule 1), ICAM-1 (intercellular adhesion molecule 1), and HIF-1α (hypoxia inducible factor 1 subunit alpha) were determined by western blot or cellular immunofluorescence. As the data showed, MEG8 was down-regulated in the carotid artery after balloon injury in rats and hypoxia-treated VSMCs, and miR-195-5p was overexpressed. Forced MEG8 overexpression or inhibiting miR-195-5p attenuated hypoxia-promoted cell proliferation and migration of VSMCs. In addition, miR-195-5p up-regulation reversed MEG8-mediated effects. Hypoxia hindered the RECK expression while boosted MMP3/9/13 levels, and the effect was markedly reversed with MEG8 up-regulation or miR-195-5p down-regulation. Mechanistically, MEG8 functioned as a competitive endogenous (ceRNA) by sponging miR-195-5p which targeted RECK. Moreover, the HIF-1α inhibitor PX478 prevented hypoxia-induced proliferation, and migration of VSMCs, upregulated MEG8, and restrained miR-195-5p expression. Overall, lncRNA MEG8 participated in hypoxia-induced excessive proliferation, inflammation and migration of VSMCs through the miR-195-5p/RECK axis.

The Role of RECK in Hepatobiliary Neoplasia Reveals Its Therapeutic Potential in NASH.

Non-alcoholic fatty liver disease (NAFLD) is a multimorbidity disorder ranging from excess accumulation of fat in the liver (steatosis) to steatohepatitis (NASH) and end-stage cirrhosis, and the development of hepatocellular carcinoma (HCC) in a subset of patients. The defining features of NASH are inflammation and progressive fibrosis. Currently, no pharmaceutical therapies are available for NAFLD, NASH and HCC; therefore, developing novel treatment strategies is desperately needed. Reversion Inducing Cysteine Rich Protein with Kazal motifs (RECK) is a well-known modifier of the extracellular matrix in hepatic remodeling and transition to HCC. More recently, its role in regulating inflammatory and fibrogenic processes has emerged. Here, we summarize the most relevant findings that extend our current understanding of RECK as a regulator of inflammation and fibrosis, and its induction as a potential strategy to blunt the development and progression of NASH and HCC.

Low RECK Expression Is Part of the Cervical Carcinogenesis Mechanisms.

Simple SummaryCervical cancer is one of the most lethal types of cancer in women from developing countries. These tumors are caused by long term infection with some human papillomavirus (HPV) types. Commonly, cervical cancer precursor lesions express high levels of matrix metalloproteinases. These enzymes break down specific components of the extracellular matrix affecting tissue structure and stiffness and cell motility. Matrix metalloproteinases and their natural inhibitors, such as Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) protein, are important in normal tissue maintenance and remodeling and play a major role in the transformation process. Here, we showed that RECK over expression reduced the tumorigenic capacity of cervical cancer cells in vivo. In addition, tumors over expressing RECK presented altered inflammatory infiltrating cells when compared to controls. Our findings are useful to further understand the biology of cervical cancer and can help to determine if RECK may be a good therapeutic target for cervical cancer treatment in the future.Human papillomavirus (HPV)-induced carcinogenesis comprises alterations in the expression and activity of matrix metalloproteinases (MMP) and their regulators. Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) inhibits the activation of specific metalloproteinases and its expression is frequently lost in human cancers. Here we analyzed the role of RECK in cervical carcinogenesis. Cervical cancer derived cell lines over expressing RECK were used to determine tumor kinetics as well as, cellular, immune and molecular properties in vivo. Besides, we analyzed RECK expression in cervical cancer samples. RECK over expression (RECK+) delayed tumor growth and increased overall survival in vivo. RECK+ tumors displayed an increase in lymphoid-like inflammatory infiltrating cells, reduced number and viability of tumor and endothelial cells and lower collagenase activity. RECK+ tumors exhibited an enrichment of cell adhesion processes both in the mouse model and cervical cancer clinical samples. Finally, we found that lower RECK mRNA levels were associated with cervical lesions progression and worse response to chemotherapy in cervical cancer patients. Altogether, we show that increased RECK expression reduced the tumorigenic potential of HPV-transformed cells both in vitro and in vivo, and that RECK down regulation is a consistent and clinically relevant event in the natural history of cervical cancer.