Specificity Protein Research Articles

KLF transcription factors in bone diseases.

Krüppel-like factors (KLFs) are crucial in the development of bone disease. They are a family of zinc finger transcription factors that are unusual in containing three highly conserved zinc finger structural domains interacting with DNA. It has been discovered that it engages in various cell functions, including proliferation, apoptosis, autophagy, stemness, invasion and migration, and is crucial for the development of human tissues. In recent years, the role of KLFs in bone physiology and pathology has received adequate attention. In addition to regulating the normal growth and development of the musculoskeletal system, KLFs participate in the pathological process of the bones and joints and are intimately linked to several skeletal illnesses, such as osteoarthritis (OA), rheumatoid arthritis (RA), osteoporosis (OP) and osteosarcoma (OS). Consequently, targeting KLFs has emerged as a promising therapeutic approach for an array of bone disorders. In this review, we summarize the current literature on the importance of KLFs in the emergence and regulation of bone illnesses, with a particular emphasis on the pertinent mechanisms by which KLFs regulate skeletal diseases. We also discuss the need for KLFs-based medication-targeted treatment. These endeavours offer new perspectives on the use of KLFs in bone disorders and provide prognostic biomarkers, therapeutic targets and possible drug candidates for bone diseases.

Silencing of circ_0088036 inhibits growth and invasion of lung adenocarcinoma through miR-203/SP1 axis.

Lung cancer is the leading cause of cancer-related deaths worldwide. Circular RNA (circRNA) circ_0088036 is a recently discovered circRNA known for its roles in rheumatoid arthritis. The study aimed to study the function of circ_0088036 in lung adenocarcinoma (LUAD).Circ_0088036 expressions were analyzed in the Gene Expression Omnibus (GEO) database. The relationship between circ_0088036 expressions and clinicopathological data of LUAD was assessed. The messenger RNAand protein levels were analyzed by quantitative real-time polymerase chain reaction and Western blot. Cell viability, apoptosis, and invasion were tested by Cell Counting Kit-8, flow cytometry, and transwell assay. The direct interaction between microRNA-203 (miR-203) and circ_0088036 or specificity protein 1 (SP1) was confirmed by dual-luciferase reporter assay, RNA pull-down, and RNA immunoprecipitation assays. Circ_0088036 was overexpressed in LUAD from the analysis of the GEO database. The poor prognosis was found in the patients with high expressions of circ_0088036. The level of Circ_0088036 was increased in LUAD tissues and cells. In terms of function, the deletion of circ_0088036 inhibited LUAD tumorigenesis in vitro by repressing cell growth, invasion, and epithelial-mesenchymal transition (EMT). In mechanism, circ_0088036 could competitively sponge miR-203, thereby affecting the expressions of the target gene SP1. In addition, lessening of miR-203 and enlarging of SP1 could eliminate the anticancer effect of short hairpin RNA-circ_0088036 on LUAD cells. Besides, the knockout of circ_0088036 hindered the growth of xenografted tumors in vivo.Circ_0088036 promoted the LUAD cell growth, invasion, and EMT via modulating the miR-203/SP1 axis in LUAD.

Abstract 3034: Krüppel-like factors in regulation of gastric cancer progression

Abstract Introduction: Gastric cancer (GC) is the third leading cause of cancer-associated mortality and the fifth most frequently diagnosed cancer worldwide. The development and progression of GC is a complicated multistage process subjected to dynamic changes in gene regulation. Investigating the effects of these changes is therefore critical for improving our knowledge about GC initiation and progression. Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with altered expressions and diverse regulatory functions in GC. We previously reported that there is loss of KLF4 in GC. Here we report that KLF4 is a crucial regulator of KLF5 expression and vice versa and together they regulate GC cell behavior. Methods: The Cancer Genome Atlas (TCGA) and pan cancer data of differential expressions of KLFs in GC was analyzed. TCGA RNAseq and clinical data for the differential expression status of KLF genes in normal and cancerous tissues and the clinicopathological association was accessed from the UALCAN web portal. The expressions of KLF4 and KLF5 in human normal stomach and GC and orthotopic xenografts were detected by immunohistochemistry (IHC). KLF4, KLF5 cross regulation was evaluated by gene manipulation of each gene individually and checking the expression of the other factor in GC cells. The effect of KLFs on GC cell proliferation, migration, and invasion was determined by Ki67 staining of tissues, PrestoBlue cell viability assay, scratch wound and transwell cell invasion assays. Results and Conclusion: Our results indicated that not only KLF4 and KLF5 have contrasting expressions and effects in GC but also these two factors cross regulate each other. While lower expressions of KLF4 was found in human GC and orthotopic xenografts, increased expression of KLF5 was found in both human GC tissue and orthotopic xenografts. KLF4 silencing and CRISPR/Cas9 knockdown in human GC cells resulted in increased expression of KLF5 which was associated with increased GC cell proliferation and invasiveness. Interestingly, KLF5 knockdown by CRISPR/Cas9 in human GC cells on the other hand resulted in increased expression of KLF4, which was associated with decreased GC cell growth. Our data therefore suggests that the differential expressions of KLF4 and KLF5 might serve as valuable prognostic markers in GC. Citation Format: Sooraj Kakkat, Prabhat Suman, Sandeep Goswami, Sujit Basu, Martin J. Heslin, Elba A. Turbat-Herrera, Veronica Ramirez Alcantara, Chandrani Sarkar, Debanjan Chakroborty. Krüppel-like factors in regulation of gastric cancer progression [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 3034.

The RNA-binding protein sorbin and SH3 domain-containing 2 are transcriptionally regulated by specificity protein 1 and function as tumor suppressors in bladder cancer by stabilizing tissue factor pathway inhibitor.

Sorbin and SH3 domain-containing 2 (SORBS2) is anRNA-binding protein and has been implicated in the development of some cancers. However, its role in bladder cancer (BC) is yet to be established. The expression of SORBS2 in BC tissues was determined from the Gene Expression Omnibusand Gene Expression Profiling Interactive Analysisdatabases and collected paired tumor/normal samples. The effects of SORBS2 on BC cells were detected by CCK-8, colony formation, Transwell, dual-luciferase, RNA immunoprecipitation, chromatin immunoprecipitation, and DNA pull-down assays. In vivo, BC cell growth and metastasis were studied by a xenograft subcutaneous model and a tail-vein metastasis model. The results showed that SORBS2 expression was significantly decreased in BC tissues and cells. SORBS2 overexpression inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transitionin vitro and tumor growth and metastasis in vivo, while silencing SORBS2 produced the opposite effect. Mechanistically, we found that SORBS2 enhanced the stability of tissue factor pathway inhibitor (TFPI) mRNA via direct binding to its 3' UTR. Restoration of TFPI expression reversed SORBS2 knockdown-induced malignant phenotypes of BC cells. In addition, SORBS2 expression was negatively regulated by the transcription factor specificity protein 1 (SP1). Conversely, SORBS2 can be transcriptionally regulated by SP1 and inhibit BC cell growth and metastasis via stabilization of TFPI mRNA, indicating SORBS2 may be a promising therapeutic target for BC.

Krüpple-like factors in cardiomyopathy: emerging player and therapeutic opportunities.

Cardiomyopathy, a heterogeneous pathological condition characterized by changes in cardiac structure or function, represents a significant risk factor for the prevalence and mortality of cardiovascular disease (CVD). Research conducted over the years has led to the modification of definition and classification of cardiomyopathy. Herein, we reviewed seven of the most common types of cardiomyopathies, including Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), diabetic cardiomyopathy, Dilated Cardiomyopathy (DCM), desmin-associated cardiomyopathy, Hypertrophic Cardiomyopathy (HCM), Ischemic Cardiomyopathy (ICM), and obesity cardiomyopathy, focusing on their definitions, epidemiology, and influencing factors. Cardiomyopathies manifest in various ways ranging from microscopic alterations in cardiomyocytes, to tissue hypoperfusion, cardiac failure, and arrhythmias caused by electrical conduction abnormalities. As pleiotropic Transcription Factors (TFs), the Krüppel-Like Factors (KLFs), a family of zinc finger proteins, are involved in regulating the setting and development of cardiomyopathies, and play critical roles in associated biological processes, including Oxidative Stress (OS), inflammatory reactions, myocardial hypertrophy and fibrosis, and cellular autophagy and apoptosis, particularly in diabetic cardiomyopathy. However, research into KLFs in cardiomyopathy is still in its early stages, and the pathophysiologic mechanisms of some KLF members in various types of cardiomyopathies remain unclear. This article reviews the roles and recent research advances in KLFs, specifically those targeting and regulating several cardiomyopathy-associated processes.

Roles of SP/KLF transcription factors in odontoblast differentiation: From development to diseases.

A zinc-finger transcription factor family comprising specificity proteins (SPs) and Krüppel-like factor proteins (KLFs) plays an important role in dentin development and regeneration. However, a systematic regulatory network involving SPs/KLFs in odontoblast differentiation has not yet been described. This review examined the expression patterns of SP/KLF gene family members and their current known functions and mechanisms in odontoblast differentiation, and discussed prospective research directions for further exploration of mechanisms involving the SP/KLF gene family in dentin development. Relevant literature on SP/KLF gene family members and dentin development was acquired from PubMed and Web of Science. We discuss the expression patterns, functions, and related mechanisms of eight members of the SP/KLF gene family in dentin development and genetic disorders with dental problems. We also summarize current knowledge about their complementary or synergistic actions. Finally, we propose future research directions for investigating the mechanisms of dentin development. The SP/KLF gene family plays a vital role in tooth development. Studying the complex complementary or synergistic interactions between SPs/KLFs is helpful for understanding the process of odontoblast differentiation. Applications of single-cell and spatial multi-omics may provide a more complete investigation of the mechanism involved in dentin development.

RBBP7, regulated by SP1, enhances the Warburg effect to facilitate the proliferation of hepatocellular carcinoma cells via PI3K/AKT signaling

BackgroundHepatocellular carcinoma (HCC) is characterized by aggressive progression and elevated mortality rates. This study aimed to investigate the regulatory effects of RBBP7 on HCC pathogenesis and the underlying mechanisms.MethodsThe expression and clinical feature of RBBP7 were evaluated using bioinformatics analysis and the assessment of clinical HCC samples. CCK8 and colony formation were employed to estimate cell proliferation function of RBBP7. Aerobic glycolysis levels of RBBP7 were evaluated by measuring ATP levels, lactic acid production, glucose uptake capacity, and the expression of relevant enzymes (PFKM, PKM2, and LDHA). The phosphorylation levels in PI3K/AKT signaling were measured by western blotting. The regulatory effect of transcription factors of specificity protein 1 (SP1) on RBBP7 mRNA expression was confirmed in dual-luciferase reporter assays and chromatin immunoprecipitation experiments. The proliferation- and glycolysis-associated proteins were assessed using immunofluorescence staining in vivo.ResultsWe found that RBBP7 is expressed at high levels in HCC and predicts poor survival. Functional assays showed that RBBP7 promoted HCC proliferation and glycolysis. Mechanistically, it was demonstrated that RBBP7 activates the PI3K/AKT pathway, a crucial pathway in glycolysis, contributing to the progression of HCC. The outcomes of the dual-luciferase assay further confirmed that SP1 is capable of activating the promoter of RBBP7.ConclusionsRBBP7, which is up-regulated by SP1, promotes HCC cell proliferation and glycolysis through the PI3K/AKT pathway. The findings of this study suggest that RBBP7 is a potential biomarker for HCC.

TET1 inhibits liver fibrosis by blocking hepatic stellate cell activation.

Hepatic stellate cells (HSCs) are critical regulator contributing to the onset and progression of liver fibrosis. Chronic liver injury triggers HSCs to undergo vast changes and trans-differentiation into a myofibroblast HSCs, the mechanism remains to be elucidated. This study investigated that the involvement of hydroxymethylase TET1 (ten-eleven translocation 1) in HSC activation and liver fibrosis. It is revealed that TET1 levels were downregulated in the livers in mouse models of liver fibrosis and patients with cirrhosis, as well as activated HSCs in comparison to quiescent HSCs. In vitro data showed that the inhibition of TET1 promoted the activation HSC, whereas TET1 overexpression inhibited HSC activation. Moreover, TET1 could regulate KLF2 (Kruppel-like transcription factors) transcription by promoting hydroxymethylation of its promoter, which in turn suppressed the activation of HSCs. In vivo, it is confirmed that liver fibrosis was aggravated in Tet1 knockout mice after CCl4 injection, accompanied by excessive activation of primary stellate cells, in contrast to wild-type mice. In conclusion, we suggested that TET1 plays a significant role in HSC activation and liver fibrosis, which provides a promising target for anti-fibrotic therapies.

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes

BackgroundVenom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris).ResultsAlthough there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis.ConclusionsOur comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

SP3-induced Timeless transcription contributes to cell growth of lung adenocarcinoma cells.

Timeless is well-known for its key role in replication checkpoints. Recent studies reveal the involvement of Timeless and specificity protein (SP) 1 in human malignancies. However, no evidence proved the interaction between SP3 and Timeless in lung adenocarcinoma (LUAD). The expression and clinical significance of Timeless were analyzed using the LUAD dataset downloaded from the Cancer Genome Atlas (TCGA). Lentivirus-mediated Timeless knockdown in A549 cells was used to examine the role of Timeless in cell proliferation and pemetrexed (PEM) resistance. Transcription factors (TFs) bound to the Timeless promoter were identified by DNA pull-down technology with HPLC-MS/MS analysis and analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Dual-luciferase reporter assay was used to determine the activity of SP3 in Timeless transcription. Timeless was overexpressed in LUAD samples, and it could serve as a potential diagnostic or prognostic biomarker for LUAD patients. shTimeless-mediated knockdown of Timeless reduced cell viability and proliferation and sensitized PEM-resistant A549 cells to PEM. Four fragments (F1: 1-373 bp), (F2: 374-962 bp), (F4: 1274-1645 bp), and (F5: 1646-2000bp) were confirmed as the TF binding profiles of the Timeless promoter. KEGG analysis showed that the TFs bound to the Timeless promoter had relevance to spliceosome, RNA transport, and mRNA surveillance pathways. SP3 promoted the transcription of Timeless via the F2 fragment (374-962 bp) binding motif. Upregulation of Timeless mediated by SP3 promotes LUAD cell proliferation, providing evidence to support that targeting the SP3/Timeless axis may be a potential therapeutic strategy against LUAD.

Transcription factor Sp1 transcriptionally enhances GSDME expression for pyroptosis

Gasdermin-E (GSDME), the executioner of pyroptosis when cleaved by caspase 3, plays a crucial role in tumor defense and the response to chemotherapy drugs in cells. So far, there are poorly known mechanisms for the expression regulation of GSDME during cell death. Here, we identify the transcription factor Sp1 (Specificity protein 1) as a positive regulator of GSDME-mediated pyroptosis. Sp1 directly interacts with the GSDME promoter at −36 ~ −28 site and promotes GSDME gene transcription. Further, Sp1 knockdown or inhibition suppresses GSDME expression, thus reducing chemotherapy drugs (topotecan, etoposide, doxorubicin, sorafinib and cisplatin) induced cell pyroptosis. The regulation process synergizes with STAT3 (Signal transducer and activator of transcription 3) activity and antagonizes with DNA methylation but barely affects GSDMD-mediated pyroptosis or TNF-induced necroptosis. Our current finding reveals a new regulating mechanism of GSDME expression, which may be a viable target for the intervention of GSDME-dependent inflammatory diseases and cancer therapy.

HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

HOOK3, a member of the human hook microtubule-tethering protein family, has been implicated in the progression of cancer. However, the role of HOOK3 in the pathogenesis of gastric cancer (GC) remains incompletely understood. In this study, we investigated the expression of HOOK3 protein in GC tissues using immunohistochemistry (IHC). The findings of our study indicate that the expression levels of HOOK3 in GC tissues were relatively low. Furthermore, a significant negative association was seen between HOOK3 expression and the prognosis of patients with GC. The suppression of HOOK3 resulted in a notable increase in the proliferation, migration, invasion, and survival of GC cells. Conversely, the overexpression of HOOK3 had the opposite impact, reducing these cellular processes. Moreover, in vivo tests have shown evidence that the overexpression of HOOK3 significantly inhibited the formation of tumors and the spread of GC cells to the lungs. In a mechanistic manner, the analysis of RNA-seq data demonstrated that the knockdown of HOOK3 resulted in a notable increase in the expression of vascular endothelial growth factor A (VEGFA) in GC cells. Furthermore, the upregulation of VEGFA counteracted the impacts of HOOK3 upregulation on the proliferation, migration, invasion, and survival of GC cells. Furthermore, it was revealed that specificity protein 1 (SP1) exhibited the ability to bind to the promoter region of VEGFA. Moreover, the overexpression of SP1 successfully counteracted the inhibitory impact of HOOK3 overexpression on the expression of VEGFA in GC cells. In summary, the results of our study indicate that HOOK3 has a role in inhibiting the growth, migration, invasion, and survival of GC cells by modulating the SP1/VEGFA pathway. These findings contribute significant knowledge to our understanding of the underlying mechanisms involved in the development of GC.

Abstract C081: The lysine demethylase KDM4C is an oncogenic driver in pancreatic ductal adenocarcinoma

Abstract Deregulation of proteins involved in chromatin regulation is common in pancreatic ductal adenocarcinoma (PDAC), occurring via both genomic and non-genomic mechanisms. Lysine demethylase 4C (KDM4C) is one of the chromatin-modifying proteins frequently overexpressed across multiple solid cancers. We observed upregulation of Kdm4c protein in a panel of human and murine PDAC cell lines, as well as in PDAC patient samples compared to non-neoplastic controls. CRISPR/Cas9-mediated deletion of KDM4C in human and murine PDAC cells resulted in reduced proliferation and clonogenicity, and increased survival of orthotopically implanted murine PDAC allografts. Notably, in multiple KDM4C-null PDAC clones analyzed by reverse phase protein arrays (RPPA), we observed downregulation of activated mitogen-activated protein kinase (MAP kinase) pathway components (including phospho-MEK and phospho-ERK), which are key effector pathways downstream of mutant KRAS. In vitro propagation of KDM4C-null PDAC lines eventually led to adaptation and restitution of MAP kinase signaling via compensatory upregulation of other KDM4 family members (KDM4A and KDM4B). In order to bypass genetic compensation by KDM4 family members, we utilized a preclinical grade pan-KDM4 inhibitor (TACH107, Tachyon Therapeutics) in PDAC cell lines, which confirmed profound reduction in proliferation and colony formation; in vivo xenograft and allograft studies with TACH107 are now ongoing. To characterize signaling pathways perturbed by KDM4C deregulation, we investigated the transcriptional changes in PDAC cells with and without KDM4C deletion by RNA-seq and integrated these with alterations in H3K9me3 and H3K36me3 marks using histone ChIP-Seq. Regulatory network analysis identified ATF4 as one of the top downregulated transcription factor networks, validating a previously published observation linking KDM4C to amino acid biosynthesis and cell proliferation. Gene set enrichment analysis showed downregulation of RAS signatures, consistent with the RPPA data on attenuation of MAP Kinase pathway. DUSP2, a member of the dual specificity protein phosphatase subfamily that inactivates ERK, is transcriptionally upregulated and has increased H3K36me3 marks near the promoter region in KDM4C-depleted cells, nominating it as a candidate of KDM4C-mediated epigenetic regulation. To further delineate the essential binding partners of KDM4C in chromatin regulation, we have created HA-tagged catalytic domain and reader domain deletion constructs that will be used for immunoprecipitation followed by mass spectrometry analysis. Overall, our data suggests that KDM4C - and other KDM4 family members – regulate MAPK signaling in KRAS-mutant cells, at least partially via attenuation of transcripts encoding specific protein phosphatases. To the best of our knowledge, this is the first demonstration linking the requirement of sustained activity of the KDM4 family of lysine demethylases to MAPK signaling in cancer and presents an opportunity to leverage this oncogenic pathway for therapeutic purposes. Citation Format: Menna-t-Allah Shaheen, Laila Barkoudeh, Sarah Dhebat, Kimal I. Rajapakshe, Bidyut Ghosh, Anirban Maitra. The lysine demethylase KDM4C is an oncogenic driver in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C081.

FSH Is Responsible for Androgen Deprivation Therapy-Associated Atherosclerosis in Mice by Exaggerating Endothelial Inflammation and Monocyte Adhesion.

Androgen deprivation therapy (ADT) is the mainstay treatment for advanced prostate cancer. But ADTs with orchiectomy and gonadotropin-releasing hormone (GnRH) agonist are associated with increased risk of cardiovascular diseases, which appears less significant with GnRH antagonist. The difference of follicle-stimulating hormone (FSH) in ADT modalities is hypothesized to be responsible for ADT-associated cardiovascular diseases. We administered orchiectomy, GnRH agonist, or GnRH antagonist in male ApoE-/- mice fed with Western diet and manipulated FSH levels by testosterone and FSH supplementation or FSH antibody to investigate the role of FSH elevation on atherosclerosis. By combining lipidomics, in vitro study, and intraluminal FSHR (FSH receptor) inhibition, we delineated the effects of FSH on endothelium and monocytes and the underlying mechanisms. Orchiectomy and GnRH agonist, but not GnRH antagonist, induced long- or short-term FSH elevation and significantly accelerated atherogenesis. In orchiectomized and testosterone-supplemented mice, FSH exposure increased atherosclerosis. In GnRH agonist-treated mice, blocking of short FSH surge by anti-FSHβ antibody greatly alleviated endothelial inflammation and delayed atherogenesis. In GnRH antagonist-treated mice, FSH supplementation aggravated atherogenesis. Mechanistically, FSH, synergizing with TNF-α (tumor necrosis factor alpha), exacerbated endothelial inflammation by elevating VCAM-1 (vascular cell adhesion protein 1) expression through the cAMP/PKA (protein kinase A)/CREB (cAMP response element-binding protein)/c-Jun and PI3K (phosphatidylinositol 3 kinase)/AKT (protein kinase B)/GSK-3β (glycogen synthase kinase 3 beta)/GATA-6 (GATA-binding protein 6) pathways. In monocytes, FSH upregulated CD29 (cluster of differentiation 29) expression via the PI3K/AKT/GSK-3β/SP1 (specificity protein 1) pathway and promoted monocyte-endothelial adhesion both in vitro and in vivo. Importantly, FSHR knockdown by shRNA in endothelium of carotid arteries markedly reduced GnRH agonist-induced endothelial inflammation and atherosclerosis in mice. FSH is responsible for ADT-associated atherosclerosis by exaggerating endothelial inflammation and promoting monocyte-endothelial adhesion.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

MiR-335 Improves Functional Recovery in Rats After Spinal Cord Injury and Protects PC12 Cells Against Injury Via the SPI-Bax/Caspase-3 Axis.

Animal and cellular models of spinal cord injury (SCI) were used to explore the role of miR-335 in regulating cell viability and apoptosis. To investigate the role and the target of miR-335 in SCI. Based on analysis of the GSE19890 data set, miR-335 was identified as a downregulated microRNA (miRNAs) following SCI. Thus, this study investigated whether downregulation of miR-335 is important in the pathological process of SCI. The GSE19890 data set investigating the expression profiles of miRNAs after SCI was downloaded from the GEO database. GSE45006 and GSE4550 data sets were used to identify differentially expressed genes between normal samples and SCI samples. The targets of rno-miR-335 were predicted using the TargetScan database.An experimental model of SCI was established, and agomir-miR-335 was intrathecally injected into rats with SCI. Functional recovery was evaluated by assessment of Basso-Beattie-Bresnahan scores and spinal cord water content and performing hematoxylin-eosin staining. Apoptosis was estimated by TUNEL staining. The H 2 O 2 -treated PC12 cells were used as in vitro models of SCI. Cell viability and apoptosis were examined by cell counting kit-8 and flow cytometry. Caspase-3 expression was evaluated by immunofluorescence staining. Levels of miRNAs and mRNAs were measured by reverse transcriptase quantitative polymerase chain reaction. Western blotting was performed to measure Bcl-2, Bax, cleaved caspase-3, and specificity protein 1 (SP1) protein levels. For in vivo studies, miR-335 was downregulated following SCI, and agomir-miR-335 delivery improved functional recovery through suppressing neuronal apoptosis by inactivating the SP1-Bax/Bcl-2/caspase-3 signaling. During in vitro analysis, miR-335 protected PC12 cells against H 2 O 2 -induced damage by negatively regulating the SP1-Bax/Bcl-2/caspase-3 signaling axis. Moreover, upregulation of SP1 abolished the apoptosis suppressive effects of miR-335 upregulation. MiR-335 ameliorates locomotor impairment in rats with SCI through the suppression of neuronal apoptosis by inactivating SP1-Bax/Bcl-2/caspase-3 signaling.

Neuron Derived Cold-Inducible RNA-Binding Protein Promotes NETs Formation to Exacerbate Brain Endothelial Barrier Disruption after Ischemic Stroke.

In ischemic stroke, neutrophils are the first-line peripheral immune cells infiltrating the brain tissue to form neutrophil extracellular traps (NETs). The present study aimed to investigate the role of neuronal cold-inducible RNA-binding protein (CIRP) in promoting NETs-induced brain endothelial barrier destruction and cerebral edema after ischemic stroke. We found that the expression of NETs and neuronal CIRP in the penumbra increased at 6 hours after transient middle cerebral artery occlusion (tMCAO) and increased significantly at 24 hours, reaching a peak at 3 days. NETs degradation or CIRP inhibition can alleviate the leakage of brain endothelial barrier and reverse the decreased expression of tight junction proteins (zonula occludens-1, claudin-5 and occludin) in tMCAO mice. Oxygen-glucose deprivation/reperfusion treated primary neurons or recombinant CIRP could induce NETs formation via TLR4/p38 signaling pathway in vitro. Transcription factor specificity protein 1 (sp1) was responsible for the increased neuronal CIRP expression and the inhibition of sp1 could suppress the increased CIRP expression, reduce NETs formation, and diminish brain endothelial barrier leakage in tMCAO mice. We also found the upregulated CIRP level was associated with severe cerebral edema in patients with acute ischemic stroke. In conclusion, the increased expression of transcription factor sp1 after ischemic stroke can lead to elevated CIRP expression and release from the neurons, which subsequently interacts with neutrophils and promotes NETs formation, resulting in brain endothelial barrier destruction and cerebral edema.

KLF6 activates Sp1-mediated prolidase transcription during TGF-β1 signaling

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline- the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors- Krüppel‐like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor β1 (TGF-β1) signaling, since both KLF6 and Sp1 are key players in TGF-β1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-β1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-β1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-β1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

Ancestral Reconstruction and the Evolution of Protein Energy Landscapes.

A protein's sequence determines its conformational energy landscape. This, in turn, determines the protein's function. Understanding the evolution of new protein functions therefore requires understanding how mutations alter the protein energy landscape. Ancestral sequence reconstruction (ASR) has proven a valuable tool for tackling this problem. In ASR, one phylogenetically infers the sequences of ancient proteins, allowing characterization of their properties. When coupled to biophysical, biochemical, and functional characterization, ASR can reveal how historical mutations altered the energy landscape of ancient proteins, allowing the evolution of enzyme activity, altered conformations, binding specificity, oligomerization, and many other protein features. In this article, we review how ASR studies have been used to dissect the evolution of energy landscapes. We also discuss ASR studies that reveal how energy landscapes have shaped protein evolution. Finally, we propose that thinking about evolution from the perspective of an energy landscape can improve how we approach and interpret ASR studies. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

MID-LIFE PLASMA PROTEINS ASSOCIATE WITH PREFRAILTY AND FRAILTY: A PROTEOMIC ANALYSIS

Abstract Physical frailty is a syndrome that typically manifests in later life, although the pathogenic process causing physical frailty likely begins decades earlier. To date, few studies have examined the biological signatures in mid-life associated with frailty in late life. We evaluated 4955 plasma proteins (log 2-transformed and standardized) measured using the SomaScan platform among 3810 participants (57.8+/-5.0 years, 58.2% women) at Visit 3 of the Atherosclerosis Risk in Community (ARIC) study, and their frailty status across up to 3 study visits in late-life (median follow-up 19 years). We used multinomial logistic regression models to examine the associations between the protein levels and the frailty status, adjusting for demographics, health indicators, comorbidities, and follow-up time. Two sets of 2 proteins were identified to be associated with prefrailty and frailty, respectively, after Bonferroni correction (p< 1.01×10-5). Higher concentration of hydroxymethylglutaryl-CoA synthase (HMGCS1; OR=1.22), a regulator of cholesterol synthesis, and lower concentration of dual specificity protein phosphatase 13 isoform A (DUSP13A; OR=0.82), a protein involved in cell apoptosis, were associated with higher odds of prefrailty. Higher concentration of growth differentiation factor 15 (GDF15; OR=1.39), a protein related to inflammation and cell senescence, and lower concentration of contactin-1 (CNTN1; OR=0.72), a protein involved in nervous system development and signalling, were associated with higher odds of frailty. The pathway and upstream regulator analyses of the physical frailty- or prefrailty-associated proteins (p< 0.01) revealed the dysregulation of apoptosis, inflammation, and neuromuscular communication in mid-life. Our findings provides new insights into frailty etiology earlier in the life course.