Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere.

Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study, we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localize to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52 to 53, localized to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four-and-half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin, and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in the maintenance and/or regulation of sarcomeric organization and function.

Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

Effects of the FHL2 gene on the development of subcutaneous and intramuscular adipocytes in goats.

BACKGROUND: Adipose tissue affects not only the meat quality of domestic animals, but also human health. Adipocyte differentiation is regulated by a series of regulatory genes and cyclins. Four and half-LIM protein (FHL2) is positively correlated with the hypertrophy of adipocytes and can cause symptoms such as obesity and diabetes. RESULT: In the transcriptome sequencing analysis of intramuscular adipocytes after three days of differentiation, the differentially expressed gene FHL2 was found. To further explore the biological significance of the differentially expressed gene FHL2, which was downregulated in the mature adipocytes. We revealed the function of FHL2 in adipogenesis through the acquisition and loss of function of FHL2. The results showed that the overexpression of FHL2 significantly increased the expression of adipogenic genes (PPARγ, C/EBPß) and the differentiation of intramuscular and subcutaneous adipocytes. However, silencing FHL2 significantly inhibited adipocyte differentiation. The overexpression of FHL2 increased the number of adipocytes stained with crystal violet and increased the mRNA expression of proliferation marker genes such as CCNE, PCNA, CCND and CDK2. In addition, it significantly increased the rate of EdU positive cells. In terms of apoptosis, overexpression of FHL2 significantly inhibited the expression of P53 and BAX in both intramuscular and subcutaneous adipocytes, which are involved in cell apoptosis. However, overexpression of FHL2 promoted the expression of BCL, but was rescued by the silencing of FHL2. CONCLUSIONS: In summary, FHL2 may be a positive regulator of intramuscular and subcutaneous adipocyte differentiation and proliferation, and acts as a negative regulator of intramuscular and subcutaneous adipocyte apoptosis. These findings provide a theoretical basis for the subsequent elucidation of FHL2 in adipocytes.

FHL2 expression by cancer-associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma.

Cancer-associated fibroblasts (CAFs) contribute to the progression of lung cancer. Four and a half LIM domain protein-2 (FHL2) is a component of focal adhesion structures. We analyzed the function of FHL2 expressed by CAFs in lung adenocarcinoma. Expression of FHL2 in fibroblast subtypes was investigated using database of single-cell RNA-sequencing of lung cancer tissue. The role of FHL2 in the proliferation and migration of CAFs was assessed. The effects of FHL2 knockout on the migration and invasion of human lung adenocarcinoma cells and tube formation of endothelial cells induced by CAF-conditioned medium (CM) were evaluated. The effect of FHL2 knockout in CAFs on metastasis was determined using a murine orthotopic lung cancer model. The prognostic significance of stromal FHL2 was assessed by immunohistochemistry in human adenocarcinoma specimens. FHL2 is highly expressed in myofibroblasts in cancer tissue. TGF-ß1 upregulated FHL2 expression in CAFs and FHL2 knockdown attenuated CAF proliferation. FHL2 knockout reduced CAF induced migration of A110L and H23 human lung adenocarcinoma cell lines, and the induction of tube formation of endothelial cells. FHL2 knockout reduced CAF-induced metastasis of lung adenocarcinomas in an orthotopic model in vivo. The concentration of Osteopontin (OPN) in CM from CAF was downregulated by FHL2 knockout. siRNA silencing and antibody blocking of OPN reduced the pro-migratory effect of CM from CAF on lung cancer cells. In resected lung adenocarcinoma specimens, positive stromal FHL2 expression was significantly associated with higher microvascular density and worse prognosis. In conclusion, FHL2 expression by CAFs enhances the progression of lung adenocarcinoma by promoting angiogenesis and metastasis.

Four and a half LIM domains 2 (FHL2) attenuates tumorigenesis of gastrointestinal stromal tumors (GISTs) by negatively regulating KIT signaling.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn't alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

Unlocking the secrets of Cardiac development and function: the critical role of FHL2.

FHL2 (Four-and-a-half LIM domain protein 2) is a crucial factor involved in cardiac morphogenesis, the process by which the heart develops its complex structure. It is expressed in various tissues during embryonic development, including the developing heart, and has been shown to play important roles in cell proliferation, differentiation, and migration. FHL2 interacts with multiple proteins to regulate cardiac development as a coactivator or a corepressor. It is involved in cardiac specification and determination of cell fate, cardiomyocyte growth, cardiac remodeling, myofibrillogenesis, and the regulation of HERG channels. Targeting FHL2 has therapeutic implications as it could improve cardiac function, control arrhythmias, alleviate heart failure, and maintain cardiac integrity in various pathological conditions. The identification of FHL2 as a signature gene in atrial fibrillation suggests its potential as a diagnostic marker and therapeutic target for this common arrhythmia.

Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial-mesenchymal transition in recurrent miscarriage.

RESEARCH QUESTION: Is four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial-mesenchymal transition (EMT) in recurrent miscarriage? DESIGN: Villus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. RESULTS: Significantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. CONCLUSIONS: FHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

FHL2 in arterial medial calcification in chronic kidney disease.

BACKGROUND AND HYPOTHESIS: Arterial medial calcification (AMC) is a common complication in individuals with chronic kidney disease (CKD), which can lead to cardiovascular morbidity and mortality. The progression of AMC is controlled by a key transcription factor called runt-related transcription factor 2 (RUNX2), which induces vascular smooth muscle cells (VSMCs) transdifferentiation into a osteogenic phenotype. However, RUNX2 has not been targeted for therapy due to its essential role in bone development. The objective of our study was to discover a RUNX2 coactivator that is highly expressed in arterial VSMCs as a potential therapy for AMC. METHODS: We employed transcriptomic analysis of human data and an animal reporter system to pinpoint FHL2 as a potential target. Subsequently, we investigated the mRNA and protein expression patterns of FHL2 in the aortas of both human and animal subjects with CKD. To examine the role of FHL2 in the RUNX2 transcription machinery, we conducted coimmunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) experiments. Next, we manipulated FHL2 expression in cultured VSMCs to examine its impact on high phosphate-induced transdifferentiation. Finally, we employed FHL2 null mice to confirm the role of FHL2 in the development of AMC in vivo. RESULTS: Among all the potential RUNX2 cofactor, FHL2 displays selective expression within the cardiovascular system. In the context of CKD subjects, FHL2 undergoes upregulation and translocation from the cytosol to the nucleus of arterial VSMCs. Once in the nucleus, FHL2 interacts structurally and functionally with RUNX2, acting as a coactivator of RUNX2. Notably, the inhibition of FHL2 expression averts transdifferentiation of VSMCs into an osteogenic phenotype and mitigates aortic calcification in uremic animals, without causing any detrimental effects on the skeletal system. CONCLUSION: These observations provide evidence that FHL2 is a promising target for treating arterial calcification in patients with CKD.

Silencing FHL2 inhibits bleomycin-induced pulmonary fibrosis through the TGF-ß1/Smad signaling pathway.

OBJECTIVE: This study aimed to investigate the inhibiting effects of FHL2 and Arbutin on cell fibrosis and their possible mechanisms. METHODS: The mRNA expression of FHL2 in pulmonary fibrosis tissues was analyzed by bioinformatics. TGF⁃ß1 induced fibrosis of mouse lung fibroblast (Mlg) and mouse primary pulmonary fibroblast (PPF) in rat's lung fibroblasts. FHL2 siRNA was transfected into Mlg and mouse PPF cells to inhibit FHL2. FHL2, α-smooth muscle actin (α-SMA), collagen 1 (Col I), and Fibronectin (Fn) were detected by qRT-PCR. Western blot expression levels of Smad3, p-Smad3, Smad2, and p-Smad2 proteins in cells. High-throughput drug screening for FHL2 inhibitors and the inhibitory effect of Arbutin on pulmonary fibrosis were validated in cellular and animal models of pulmonary fibrosis. RESULTS: The mRNA expression of FHL2 in lung fiber tissue was increased. Meanwhile, the decrease of FHL2 expression significantly inhibited the cellular fibrosis morphological changes of rat's lung fibroblasts (Mlgs) and primary lung fibroblasts (PPFs). The expression levels of α⁃SMA, Col I, and Fn were decreased. High-throughput screening showed that Arbutin targeted FHL2. Arbutin alleviated bleomycin (BLM)-induced pulmonary fibrosis in rats by inhibiting FHL2 and then the TGF-ß1/Smad signaling pathway. CONCLUSION: Inhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃ß1 and bleomycin, and then inhibit the fibrosis.

Hsa-miR-92b-3p Targeting FHL2 to Enhance Radiosensitivity of Nasopharyngeal Carcinoma.

Radiotherapy resistance is a major cause of treatment failure and leads to poor prognosis in nasopharyngeal carcinoma (NPC). Evidences indicate that microRNA (miRNAs) are closely associated with radiotherapy for NPC. In this study, we found that the expression level of miR-92b-3p was significantly higher in radiotherapy-sensitive NPC patients than in radiotherapy-resistant patients. High expression of miR-92b-3p was associated with good prognosis in patients with NPC, and high expression of FHL2 was associated with poor prognosis in patients with NPC. It was predicted that miR-92b-3p could directly target and bind FHL2. Overexpression of miR-92b-3p significantly inhibited FHL2 expression at the mRNA as well as protein levels, while inhibition of miR-92b-3p expression significantly upregulated FHL2 expression. Overexpression of miR-92b-3p significantly reduced proliferation and colony formation in NPC cells. Inhibition of miR-92b-3p attenuated the sensitivity of nasopharyngeal carcinoma to radiotherapy, while simultaneous inhibition of miR-92b-3p and FHL2 increased the sensitivity of NPC to radiotherapy. Our findings highlighted that miR-92b-3p is closely associated with radiotherapy sensitivity and prognosis in NPC patients and may improve the sensitivity of NPC to radiotherapy by targeting FHL2.

Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

FHL2 Genetic Polymorphisms and Pro-Diabetogenic Lipid Profile in the Multiethnic HELIUS Cohort.

Type 2 diabetes mellitus (T2D) is a prevalent disease often accompanied by the occurrence of dyslipidemia. Four and a half LIM domains 2 (FHL2) is a scaffolding protein, whose involvement in metabolic disease has recently been demonstrated. The association of human FHL2 with T2D and dyslipidemia in a multiethnic setting is unknown. Therefore, we used the large multiethnic Amsterdam-based Healthy Life in an Urban Setting (HELIUS) cohort to investigate FHL2 genetic loci and their potential role in T2D and dyslipidemia. Baseline data of 10,056 participants from the HELIUS study were available for analysis. The HELIUS study contained individuals of European Dutch, South Asian Surinamese, African Surinamese, Ghanaian, Turkish, and Moroccan descent living in Amsterdam and were randomly sampled from the municipality register. Nineteen FHL2 polymorphisms were genotyped, and associations with lipid panels and T2D status were investigated. We observed that seven FHL2 polymorphisms associated nominally with a pro-diabetogenic lipid profile including triglyceride (TG), high-density and low-density lipoprotein-cholesterol (HDL-C and LDL-C), and total cholesterol (TC) concentrations, but not with blood glucose concentrations or T2D status in the complete HELIUS cohort upon correcting for age, gender, BMI, and ancestry. Upon stratifying for ethnicity, we observed that only two of the nominally significant associations passed multiple testing adjustments, namely, the association of rs4640402 with increased TG and rs880427 with decreased HDL-C concentrations in the Ghanaian population. Our results highlight the effect of ethnicity on pro-diabetogenic selected lipid biomarkers within the HELIUS cohort, as well as the need for more large multiethnic cohort studies.

FHL2 Inhibits SARS-CoV-2 Replication by Enhancing IFN-ß Expression through Regulating IRF-3.

SARS-CoV-2 triggered the global COVID-19 pandemic, posing a severe threat to public health worldwide. The innate immune response in cells infected by SARS-CoV-2 is primarily orchestrated by type I interferon (IFN), with IFN-ß exhibiting a notable inhibitory impact on SARS-CoV-2 replication. FHL2, acting as a docking site, facilitates the assembly of multiprotein complexes and regulates the transcription of diverse genes. However, the association between SARS-CoV-2 and FHL2 remains unclear. In this study, we report for the first time that SARS-CoV-2 infection in Caco2 cells results in the upregulation of FHL2 expression, while the virus's N proteins can enhance FHL2 expression. Notably, the knockdown of FHL2 significantly amplifies SARS-CoV-2 replication in vitro. Conversely, the overexpression of FHL2 leads to a marked reduction in SARS-CoV-2 replication, with the antiviral property of FHL2 being independent of the cell or virus type. Subsequent experiments reveal that FHL2 supports IFN-ß transcription by upregulating the expression and phosphorylation of IRF-3, thereby impeding SARS-CoV-2 replication in cells. These findings highlight FHL2 as a potential antiviral target for treating SARS-CoV-2 infections.

Glucose-mediated insulin secretion is improved in FHL2-deficient mice and elevated FHL2 expression in humans is associated with type 2 diabetes.

AIMS/HYPOTHESIS: The general population is ageing, involving an enhanced incidence of chronic diseases such as type 2 diabetes. With ageing, DNA methylation of FHL2 increases, as well as expression of the four and a half LIM domains 2 (FHL2) protein in human pancreatic islets. We hypothesised that FHL2 is actively involved in glucose metabolism. METHODS: Publicly available microarray datasets from human pancreatic islets were analysed for FHL2 expression. In FHL2-deficient mice, we studied glucose clearance and insulin secretion. Gene expression analysis and glucose-stimulated insulin secretion (GSIS) were determined in isolated murine FHL2-deficient islets to evaluate insulin-secretory capacity. Moreover, knockdown and overexpression of FHL2 were accomplished in MIN6 cells to delineate the underlying mechanism of FHL2 function. RESULTS: Transcriptomics of human pancreatic islets revealed that individuals with elevated levels of HbA1c displayed increased FHL2 expression, which correlated negatively with insulin secretion pathways. In line with this observation, FHL2-deficient mice cleared glucose more efficiently than wild-type littermates through increased plasma insulin levels. Insulin sensitivity was comparable between these genotypes. Interestingly, pancreatic islets isolated from FHL2-deficient mice secreted more insulin in GSIS assays than wild-type mouse islets even though insulin content and islet size was similar. To support this observation, we demonstrated increased expression of the transcription factor crucial in insulin secretion, MAF BZIP transcription factor A (MafA), higher expression of GLUT2 and reduced expression of the adverse factor c-Jun in FHL2-deficient islets. The underlying mechanism of FHL2 was further delineated in MIN6 cells. FHL2-knockdown led to enhanced activation of forkhead box protein O1 (FOXO1) and its downstream genes such as Mafa and Pdx1 (encoding pancreatic and duodenal homeobox 1), as well as increased glucose uptake. On the other hand, FHL2 overexpression in MIN6 cells blocked GSIS, increased the formation of reactive oxygen species and increased c-Jun activity. CONCLUSIONS/INTERPRETATION: Our data demonstrate that FHL2 deficiency improves insulin secretion from beta cells and improves glucose tolerance in mice. Given that FHL2 expression in humans increases with age and that high expression levels of FHL2 are associated with beta cell dysfunction, we propose that enhanced FHL2 expression in elderly individuals contributes to glucose intolerance and the development of type 2 diabetes. DATA AVAILABILITY: The human islet microarray datasets used are publicly available and can be found on https://www.ncbi.nlm.nih.gov/geo/ .

High level of FHL2 exacerbates the outcome of non-small cell lung cancer (NSCLC) patients and the malignant phenotype in NSCLC cells.

Non-small cell lung cancer (NSCLC) is a malignant tumour with high mortality. FHL2 has been identified as a biomarker of lung cancer. This research explored the effects of FHL2 expression on NSCLC. NSCLC-associated data sets were collected from the assistant for clinical bioinformatics and TCGA databases respectively. The association between FHL2 and clinical characteristics, the prognostic significance of FHL2 and the influences of various variables on NSCLC were determined by Pearson's chi-squared test, the Kaplan-Meier curve and the Cox regression model respectively. FHL2 level was altered by cell transfection and was measured by qRT-PCR. Tumour xenograft formation was completed by inoculating sh-FHL2/pcDNA-FHL2 transfected cells into BALB/c nude mice. Protein expression was assessed by western blot. Cell apoptosis, proliferation and epithelial - mesenchymal transition (EMT) characteristics were evaluated employing TUNEL, BrdU+ and microscopic observation respectively. The expression of Ki67 and N-cadherin was assessed by immunohistochemistry. The results showed that FHL2 was highly expressed in NSCLC tissues. Patients with high FHL2 expression experienced lower overall survival probability. FHL2 knockdown promoted apoptosis, but inhibited EMT of A549 and NCI-H460 cells, which was verified by the increased ratios of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3, as well as augmented E-cadherin and reduced N-cadherin. In an in vivo assay FHL2 knockdown decreased tumour volume and weight, repressed EMT, but enhanced apoptosis. FHL2 upregulation showed the opposite effects of FHL2 knockdown. Furthermore, FHL2 upregulation facilitated cell proliferation both in in vitro and in vivo assays. These outcomes indicated that high level of FHL2 facilitated tumorigenesis, as well as the proliferation and EMT of NSCLC cells.

Screening and identification of a novel FHL2 mutation by whole exome sequencing in twins with familial Waldenström macroglobulinemia.

BACKGROUND: Waldenström macroglobulinemia (WM) is a rare chronic B-cell lymphoma. Familial clustering of WM has been observed over the years. However, little is known about the contribution of inherited genetic variants to familial WM cases. METHODS: The authors performed whole exome sequencing (WES) of germline DNA samples from twins, one diagnosed with WM and the other diagnosed with immunoglobulin M monoclonal gammopathy of undetermined significance, and their healthy siblings. Bioinformatics analysis of public biological databases was used to identify the most relevant familial WM candidate from WES. Transcript expression and protein levels of the familial WM candidate were evaluated in the WM patient and 2 unaffected members of the kindred. RESULTS: Among the 10 shared candidate mutations in the twins, the authors identified a novel heterozygous germline mutation in four and a half LIM domains protein 2 (FHL2; c.G226A, p.V76M) as a familial WM-associated mutation. FHL2 appeared to be connected with reported signaling pathways and disease-driving genes such as IL6 and HCK in WM. In addition, the authors found reduced FHL2 messenger RNA and protein expression in peripheral blood samples from the patient with WM in comparison with the healthy siblings. CONCLUSIONS: Taken together, these findings indicate that an FHL2g226a mutation may play an important role in familial WM, and they provide new screening possibilities for familial cases. LAY SUMMARY: Familial clustering in Waldenström macroglobulinemia (WM) has been observed over the years. The authors performed whole exome sequencing of germline DNA samples from twins, one diagnosed with WM and the other diagnosed with immunoglobulin M monoclonal gammopathy of undetermined significance, and their healthy siblings. Among the 10 shared candidate mutations in the twins, a novel heterozygous germline mutation in four and a half LIM domains protein 2 (FHL2; c.G226A, p.V76M) was identified as the most relevant familial WM candidate through bioinformatics analysis of a public database. Also, messenger RNA and protein expression of FHL2 was significantly lower in peripheral blood mononuclear cells of the WM patient in comparison with the healthy siblings, and this suggested that the function of FHL2 was impaired when mutated.

Deficiency of Fhl2 leads to delayed neuronal cell migration and premature astrocyte differentiation.

The four and a half LIM domains protein 2 (Fhl2) is an adaptor protein capable of mediating protein-protein interactions. Here, we report for the first time phenotypic changes in the brain of Fhl2-deficient mice. We showed that Fhl2 is expressed in neural stem cells, precursors and mature cells of neuronal lineage. Moreover, Fhl2 deficiency leads to delayed neuroblast migration in vivo, premature astroglial differentiation of neural stem cells (NSCs) in vitro, and a gliosis-like accumulation of glial fibrillary acidic protein (GFAP)-positive astrocytes in vivo that substantially increases with age. Collectively, Fhl2-deficiency in the brain interrupts the maintenance and the balanced differentiation of adult NSCs, resulting in preferentially glial differentiation and early exhaustion of the NSC pool required for adult neurogenesis.

Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein.

Decades of insufficient control have resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe, and millions have suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with the hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a prerequisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has a detectable stimulatory effect(s) on CHIKV replication, and Fhl1 knockout (KO) cell lines demonstrate slower infection spread. Nuclear magnetic resonance (NMR)-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps that of another proviral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by the LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP-binding sites remain viable and develop spreading infection in multiple cell types. Our study also demonstrated that other members of the FHL family can bind to CHIKV HVD and thus may be involved in viral replication.IMPORTANCE Replication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of the cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and, subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive proviral functions. Elimination of the FHL1-binding site in the nsP3 HVD can be used for the development of stable, attenuated vaccine candidates.

Identifying Methylation Patterns in Dental Pulp Aging: Application to Age-at-Death Estimation in Forensic Anthropology.

Age-at-death estimation constitutes one of the key parameters for identification of human remains in forensic investigations. However, for applications in forensic anthropology, many current methods are not sufficiently accurate for adult individuals, leading to chronological age estimates erring by ±10 years. Based on recent trends in aging studies, DNA methylation has great potential as a solution to this problem. However, there are only a few studies that have been published utilizing DNA methylation to determine age from human remains. The aim of the present study was to expand the range of this work by analyzing DNA methylation in dental pulp from adult individuals. Healthy erupted third molars were extracted from individuals aged 22-70. DNA from pulp was isolated and bisulfite converted. Pyrosequencing was the chosen technique to assess DNA methylation. As noted in previous studies, we found that ELOVL2 and FHL2 CpGs played a role in age estimation. In addition, three new markers were evaluated-NPTX2, KLF14, and SCGN. A set of CpGs from these five loci was used in four different multivariate regression models, providing a Mean Absolute Error (MAE) between predicted and chronological age of 1.5-2.13 years. The findings from this research can improve age estimation, increasing the accuracy of identification in forensic anthropology.

Deletion of the FHL2 gene attenuates intima-media thickening in a partially ligated carotid artery ligated mouse model.

The four and a half LIM domain protein 2 (FHL2) is a member of the four and a half LIM domain (FHL) gene family, and it is associated with cholesterol-enriched diet-promoted atherosclerosis. However, the effect of FHL2 protein on vascular remodelling in response to hemodynamic alterations remains unclear. Here, we investigated the role of FHL2 in a model of restricted blood flow-induced atherosclerosis. To promote neointimal hyperplasia in vivo, we subjected FHL2+/+ and FHL2-/- mice to partial ligation of the left carotid artery (LCA). The expression of p-ERK and p-AKT was decreased in FHL2-/- mice. FHL2 bound to AKT regulated AKT phosphorylation and led to Rac1-GTP inactivation. FHL2 silencing in human aortic smooth muscle cells down-regulated the PDGF-induced phosphorylation of ERK and AKT. Furthermore, FHL2 silencing reduced cytoskeleton conformational changes and caused cell cycle arrest. We concluded that FHL2 is essential for the regulation of arterial smooth muscle cell function. FHL2 modulates proliferation and migration via mitogen-activated protein kinase (MAPK) and PI3K-AKT signalling, leading to arterial wall thickening and thus neointimal hyperplasia.