A mouse model of Zhu-Tokita-Takenouchi-Kim syndrome reveals indispensable SON functions in organ development and hematopoiesis.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss of function of SON. While patients with ZTTK syndrome live with numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifted cell fate more toward the myeloid lineage but compromised lymphoid lineage development by reducing genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency caused inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

SON is an essential m6A target for hematopoietic stem cell fate.

Stem cells regulate their self-renewal and differentiation fate outcomes through both symmetric and asymmetric divisions. m6A RNA methylation controls symmetric commitment and inflammation of hematopoietic stem cells (HSCs) through unknown mechanisms. Here, we demonstrate that the nuclear speckle protein SON is an essential m6A target required for murine HSC self-renewal, symmetric commitment, and inflammation control. Global profiling of m6A identified that m6A mRNA methylation of Son increases during HSC commitment. Upon m6A depletion, Son mRNA increases, but its protein is depleted. Reintroduction of SON rescues defects in HSC symmetric commitment divisions and engraftment. Conversely, Son deletion results in a loss of HSC fitness, while overexpression of SON improves mouse and human HSC engraftment potential by increasing quiescence. Mechanistically, we found that SON rescues MYC and suppresses the METTL3-HSC inflammatory gene expression program, including CCL5, through transcriptional regulation. Thus, our findings define a m6A-SON-CCL5 axis that controls inflammation and HSC fate.

A mouse model of ZTTK syndrome reveals indispensable SON functions in organ development and hematopoiesis.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hamper our understanding of both SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing key genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in organ development and hematopoiesis. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

Nuclear FAK in endothelium: An intrinsic inhibitor of NF-κB activation in atherosclerosis.

BACKGROUND AND AIMS: Hyperlipidemia leads to the accumulation of oxidized low-density lipoprotein (oxLDL) within the vessel wall where it causes chronic inflammation in endothelial cells (ECs) and drives atherosclerotic lesions. Although focal adhesion kinase (FAK) is critical in proinflammatory NF-κB activation in ECs, it is unknown if hyperlipidemia alters FAK-mediated NF-κB activity in vivo to affect atherosclerosis progression. METHODS: We investigated changes in EC FAK and NF-κB activation using Apoe-/- mice fed a western diet (WD). Both pharmacological FAK inhibition and EC-specific FAK inhibited mouse models were utilized. FAK and NF-κB localization and activity were also analyzed in human atherosclerotic samples. RESULTS: ECs of hyperlipidemic mice clearly showed much higher levels of FAK activation in the cytoplasm, which was associated with increased NF-κB activation compared to normal diet (ND) group. On the contrary, FAK is mostly localized in the nucleus and inactive in ECs under healthy conditions with a low NF-κB activity. Both pharmacological and EC-specific genetic FAK inhibition in WD fed Apoe-/- mice exhibited a significant decrease in FAK activity and cytoplasmic localization, NF-κB activation, macrophage recruitment, and atherosclerotic lesions compared to the vehicle or FAK wild-type groups. Analyses of human atherosclerotic specimens revealed a positive correlation between increased active cytoplasmic FAK within ECs and NF-κB activation in the lesions. CONCLUSIONS: Hyperlipidemic conditions activate NF-κB pathway by increasing EC FAK activity and cytoplasmic localization in mice and human atherosclerotic samples. As FAK inhibition can efficiently reduce vascular inflammation and atherosclerotic lesions in mice by reversing EC FAK localization and NF-κB activation, these findings support a potential use for FAK inhibitors in treating atherosclerosis.

SON-Related Zhu-Tokita-Takenouchi-Kim Syndrome With Recurrent Hemiplegic Migraine: Putative Role of PRRT2.

Background and Objectives: Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome (OMIM 617140) is a recently identified neurodevelopmental disorder caused by heterozygous loss-of-function (LoF) variants in SON. Because the SON protein functions as an RNA-splicing regulator, it has been shown that some clinical features of ZTTK syndrome can be attributed to abnormal RNA splicing. Several neurologic features have been observed in patients with ZTTK syndrome, including seizure/epilepsy and other EEG abnormalities. However, a relationship between SON LoF in ZTTK syndrome and hemiplegic migraine remains unknown. Methods: We identified a patient with a pathogenic variant in SON who shows typical clinical features of ZTTK syndrome and experienced recurrent episodes of hemiplegic migraine. To define clinical features, brain MRI and EEG during and after episodes of hemiplegic migraine were characterized. To identify molecular mechanisms for this clinical presentation, we investigated the impact of small interfering RNA (siRNA)-mediated SON knockdown on mRNA expression of the CACNA1A, ATP1A2, SCN1A, and PRRT2 genes, known to be associated with hemiplegic migraine, by quantitative RT-PCR. Pre-mRNA splicing of PRRT2 on SON knockdown was further examined by RT-PCR using primers targeting specific exons. Results: Recurrent episodes of hemiplegic migraine in our patient typically followed modest closed head injuries, and recurrent seizures occurred during the most severe of these episodes. Transient hemispheric cortical interstitial edema and asymmetric EEG slowing were identified during episodes. Our siRNA experiments revealed that SON knockdown significantly reduces PRRT2 mRNA levels in U87MG and SH-SY5Y cell lines, although a reduction in CACNA1A, ATP1A2, and SCN1A mRNA expression was not observed. We further identified that SON knockdown leads to failure in intron 2 removal from PRRT2 pre-mRNA, resulting in a premature termination codon that blocks the generation of functionally intact full-length PRRT2. Discussion: This report identifies recurrent hemiplegic migraine as a novel clinical manifestation of ZTTK syndrome, further characterizes this clinical feature, and provides evidence for downregulation of PRRT2 caused by SON LoF as a mechanism causing hemiplegic migraine. Examination of the SON gene may be indicated in individuals with recurrent hemiplegic migraine.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation.

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor-induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

AIMS: Vascular smooth muscle cells (VSMCs) normally exhibit a very low proliferative rate. Vessel injury triggers VSMC proliferation, in part, through focal adhesion kinase (FAK) activation, which increases transcription of cyclin D1, a key activator for cell cycle-dependent kinases (CDKs). At the same time, we also observe that FAK regulates the expression of the CDK inhibitors (CDKIs) p27 and p21. However, the mechanism of how FAK controls CDKIs in cell cycle progression is not fully understood. METHODS AND RESULTS: We found that pharmacological and genetic FAK inhibition increased p27 and p21 by reducing stability of S-phase kinase-associated protein 2 (Skp2), which targets theCDKIs for degradation. FAK N-terminal domain interacts with Skp2 and an APC/C E3 ligase activator fizzy-related 1 (Fzr1) in the nucleus, which promote ubiquitination and degradation of both Skp2 and Fzr1. Notably, overexpression of cyclin D1 alone failed to promote proliferation of genetic FAK kinase-dead (KD) VSMCs, suggesting that the FAK-Skp2-CDKI signalling axis is distinct from the FAK-cyclin D1 pathway. However, overexpression of both cyclin D1 and Skp2 enabled proliferation of FAK-KD VSMCs, implicating that FAK ought to control both activating and inhibitory switches for CDKs. In vivo, wire injury activated FAK in the cytosol, which increased Skp2 and decreased p27 and p21 levels. CONCLUSION: Both pharmacological FAK and genetic FAK inhibition reduced Skp2 expression in VSMCs upon injury, which significantly reduced intimal hyperplasia through elevated expression of p27 and p21. This study revealed that nuclear FAK-Skp2-CDKI signalling negatively regulates CDK activity in VSMC proliferation.

Establishing the phenotypic spectrum of ZTTK syndrome by analysis of 52 individuals with variants in SON.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, an intellectual disability syndrome first described in 2016, is caused by heterozygous loss-of-function variants in SON. Its encoded protein promotes pre-mRNA splicing of many genes essential for development. Whereas individual phenotypic traits have previously been linked to erroneous splicing of SON target genes, the phenotypic spectrum and the pathogenicity of missense variants have not been further evaluated. We present the phenotypic abnormalities in 52 individuals, including 17 individuals who have not been reported before. In total, loss-of-function variants were detected in 49 individuals (de novo in 47, inheritance unknown in 2), and in 3, a missense variant was observed (2 de novo, 1 inheritance unknown). Phenotypic abnormalities, systematically collected and analyzed in Human Phenotype Ontology, were found in all organ systems. Significant inter-individual phenotypic variability was observed, even in individuals with the same recurrent variant (n = 13). SON haploinsufficiency was previously shown to lead to downregulation of downstream genes, contributing to specific phenotypic features. Similar functional analysis for one missense variant, however, suggests a different mechanism than for heterozygous loss-of-function. Although small in numbers and while pathogenicity of these variants is not certain, these data allow for speculation whether de novo missense variants cause ZTTK syndrome via another mechanism, or a separate overlapping syndrome. In conclusion, heterozygous loss-of-function variants in SON define a recognizable syndrome, ZTTK, associated with a broad, severe phenotypic spectrum, characterized by a large inter-individual variability. These observations provide essential information for affected individuals, parents, and healthcare professionals to ensure appropriate clinical management.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

SON drives oncogenic RNA splicing in glioblastoma by regulating PTBP1/PTBP2 switching and RBFOX2 activity.

While dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors.

eNEMAL, an enhancer RNA transcribed from a distal MALAT1 enhancer, promotes NEAT1 long isoform expression.

Emerging evidence has shown that active enhancers are abundantly transcribed, generating long non-coding RNAs, called enhancer RNAs (eRNAs). While putative eRNAs are often observed from RNA sequencing, the roles of most eRNAs remain largely unknown. Previously, we identified putative enhancer regions at the MALAT1 locus that form chromatin-chromatin interactions under hypoxia, and one of these enhancers is located about 30 kb downstream of the NEAT1 gene and -20 kb upstream of the MALAT1 gene (MALAT1-20 kb enhancer). Here, we report that a novel eRNA, named eRNA of the NEAT1-MALAT1-Locus (eNEMAL), is transcribed from the MALAT1-20 kb enhancer and conserved in primates. We found that eNEMAL is upregulated in response to hypoxia in multiple breast cancer cell lines, but not in non-tumorigenic MCF10A cells. Overexpression and knockdown of eNEMAL revealed that alteration of eNEMAL level does not affect MALAT1 expression. Instead, we found that eNEMAL upregulates the long isoform of NEAT1 (NEAT1_2) without increasing the total NEAT1 transcript level in MCF7 breast cancer cells, suggesting that eNEMAL has a repressive effect on the 3'-end polyadenylation process required for generating the short isoform of NEAT1 (NEAT1_1). Altogether, we demonstrated that an eRNA transcribed from a MALAT1 enhancer regulates NEAT1 isoform expression, implicating the MALAT1-20 kb enhancer and its transcript eNEMAL in co-regulation of MALAT1 and NEAT1 in response to hypoxia in breast cancer cells.

son is necessary for proper vertebrate blood development.

The gene SON is on human chromosome 21 (21q22.11) and is thought to be associated with hematopoietic disorders that accompany Down syndrome. Additionally, SON is an RNA splicing factor that plays a role in the transcription of leukemia-associated genes. Previously, we showed that mutations in SON cause malformations in human and zebrafish spines and brains during early embryonic development. To examine the role of SON in normal hematopoiesis, we reduced expression of the zebrafish homolog of SON in zebrafish at the single-cell developmental stage with specific morpholinos. In addition to the brain and spinal malformations we also observed abnormal blood cell levels upon son knockdown. We then investigated how blood production was altered when levels of son were reduced. Decreased levels of son resulted in lower amounts of red blood cells when visualized with lcr:GFP transgenic fish. There were also reduced thrombocytes seen with cd41:GFP fish, and myeloid cells when mpx:GFP fish were examined. We also observed a significant decrease in the quantity of T cells, visualized with lck:GFP fish. However, when we examined their hematopoietic stem and progenitor cells (HSPCs), we saw no difference in colony-forming capability. These studies indicate that son is essential for the proper differentiation of the innate and adaptive immune system, and further investigation determining the molecular pathways involved during blood development should elucidate important information about vertebrate HSPC generation, proliferation, and differentiation.

Focal Adhesion Kinase Activity and Localization is Critical for TNF-α-Induced Nuclear Factor-κB Activation.

While sustained nuclear factor-κB (NF-κB) activation is critical for proinflammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases.

SON inhibits megakaryocytic differentiation via repressing RUNX1 and the megakaryocytic gene expression program in acute megakaryoblastic leukemia.

A high incidence of acute megakaryoblastic leukemia (AMKL) in Down syndrome patients implies that chromosome 21 genes have a pivotal role in AMKL development, but the functional contribution of individual genes remains elusive. Here, we report that SON, a chromosome 21-encoded DNA- and RNA-binding protein, inhibits megakaryocytic differentiation by suppressing RUNX1 and the megakaryocytic gene expression program. As megakaryocytic progenitors differentiate, SON expression is drastically reduced, with mature megakaryocytes having the lowest levels. In contrast, AMKL cells express an aberrantly high level of SON, and knockdown of SON induced the onset of megakaryocytic differentiation in AMKL cell lines. Genome-wide transcriptome analyses revealed that SON knockdown turns on the expression of pro-megakaryocytic genes while reducing erythroid gene expression. Mechanistically, SON represses RUNX1 expression by directly binding to the proximal promoter and two enhancer regions, the known +23 kb enhancer and the novel +139 kb enhancer, at the RUNX1 locus to suppress H3K4 methylation. In addition, SON represses the expression of the AP-1 complex subunits JUN, JUNB, and FOSB which are required for late megakaryocytic gene expression. Our findings define SON as a negative regulator of RUNX1 and megakaryocytic differentiation, implicating SON overexpression in impaired differentiation during AMKL development.

Targeting focal adhesion kinase in cancer cells and the tumor microenvironment.

Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that is frequently overexpressed in advanced human cancers. Recent studies have demonstrated that aside from FAK's catalytic activity in cancer cells, its cellular localization is also critical for regulating the transcription of chemokines that promote a favorable tumor microenvironment (TME) by suppressing destructive host immunity. In addition to the protumor roles of FAK in cancer cells, FAK activity within cells of the TME may also support tumor growth and metastasis through various mechanisms, including increased angiogenesis and vascular permeability and effects related to fibrosis in the stroma. Small molecule FAK inhibitors have demonstrated efficacy in alleviating tumor growth and metastasis, and some are currently in clinical development phases. However, several preclinical trials have shown increased benefits from dual therapies using FAK inhibitors in combination with other chemotherapies or with immune cell activators. This review will discuss the role of nuclear FAK as a driver for tumor cell survival as well as potential therapeutic strategies to target FAK in both tumors and the TME.

Hypoxia induces cancer cell-specific chromatin interactions and increases MALAT1 expression in breast cancer cells.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA overexpressed in various cancers that promotes cell growth and metastasis. Although hypoxia has been shown to up-regulate MALAT1, only hypoxia-inducible factors (HIFs) have been implicated in activation of the MALAT1 promoter in specific cell types and other molecular mechanisms associated with hypoxia-mediated MALAT1 up-regulation remain largely unknown. Here, we demonstrate that hypoxia induces cancer cell-specific chromatin-chromatin interactions between newly identified enhancer-like cis-regulatory elements present at the MALAT1 locus. We show that hypoxia-mediated up-regulation of MALAT1 as well as its antisense strand TALAM1 occurs in breast cancer cells, but not in nontumorigenic mammary epithelial cells. Our analyses on the MALAT1 genomic locus discovered three novel putative enhancers that are located upstream and downstream of the MALAT1 gene body. We found that parts of these putative enhancers are epigenetically modified to a more open chromatin state under hypoxia in breast cancer cells. Furthermore, our chromosome conformation capture experiment demonstrated that noncancerous cells and breast cancer cells exhibit different interaction profiles under both normoxia and hypoxia, and only breast cancer cells gain specific chromatin interactions under hypoxia. Although the HIF-2α protein can enhance the interaction between the promoter and the putative 3' enhancer, the gain of chromatin interactions associated with other upstream elements, such as putative -7 and -20 kb enhancers, were HIF-independent events. Collectively, our study demonstrates that cancer cell-specific chromatin-chromatin interactions are formed at the MALAT1 locus under hypoxia, implicating a novel mechanism of MALAT1 regulation in cancer.

FAK and Pyk2 activity promote TNF-α and IL-1ß-mediated pro-inflammatory gene expression and vascular inflammation.

Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1ß (IL-1ß) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1ß stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1ß signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE-/- mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.

Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription.

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.

SON haploinsufficiency causes impaired pre-mRNA splicing of CAKUT genes and heterogeneous renal phenotypes.

Although genetic testing is increasingly used in clinical nephrology, a large number of patients with congenital abnormalities of the kidney and urinary tract (CAKUT) remain undiagnosed with current gene panels. Therefore, careful curation of novel genetic findings is key to improving diagnostic yields. We recently described a novel intellectual disability syndrome caused by de novo heterozygous loss-of-function mutations in the gene encoding the splicing factor SON. Here, we show that many of these patients, including two previously unreported, exhibit a wide array of kidney abnormalities. Detailed phenotyping of 14 patients with SON haploinsufficiency identified kidney anomalies in 8 patients, including horseshoe kidney, unilateral renal hypoplasia, and renal cysts. Recurrent urinary tract infections, electrolyte disturbances, and hypertension were also observed in some patients. SON knockdown in kidney cell lines leads to abnormal pre-mRNA splicing, resulting in decreased expression of several established CAKUT genes. Furthermore, these molecular events were observed in patient-derived cells with SON haploinsufficiency. Taken together, our data suggest that the wide spectrum of phenotypes in patients with a pathogenic SON mutation is a consequence of impaired pre-mRNA splicing of several CAKUT genes. We propose that genetic testing panels designed to diagnose children with a kidney phenotype should include the SON gene.

FAK inhibition reduces metastasis of α4 integrin-expressing melanoma to lymph nodes by targeting lymphatic VCAM-1 expression.

Malignant melanoma typically metastasizes to lymph nodes (LNs) as a primary or in-transit lesion before secondary metastasis occurs, and LN biopsy is a common procedure to diagnose melanoma progression. Since cancer metastasis is a complex process where various interactions between tumor cells and the stroma play key roles in establishing metastatic lesions, the exact mechanisms underlying melanoma metastasis to LNs remains unknown. It has been known that focal adhesion kinase (FAK) activity promotes the expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1). As VCAM-1 is a major receptor for α4 integrin and plays a key role in leukocyte recruitment, we reasoned that inhibition of FAK activity may reduce VCAM-1 expression within LNs and thus reduce metastasis of α4 integrin-expressing melanoma to LNs. First, we found that a pharmacological FAK inhibitor, PF-271, blocked tumor necrosis factor-α (TNF-α)-mediated VCAM-1 expression on human dermal lymphatic endothelial cells (HDLECs). In vitro, PF-271 significantly decreased B16F10 melanoma adhesion to and transmigration through HDLECs compared to TNF-α treated cells. Furthermore, in vivo FAK inhibition by oral PF-271 administration reduced VCAM-1 expression in inguinal, cervical, and popliteal LNs compared to vehicle treated mice. Finally, in a footpad metastasis model, B16F10 melanoma cells were injected into the right footpad of C57BL/6 mice, and PF-271 (50 mg/kg, twice daily for 6 days) was orally administrated after 1 week of tumor transplantation. While untreated mice exhibited significant metastatic melanoma lesions in popliteal LNs, PF-271 treated mice showed only marginal melanoma metastasis. These results support the possibility that FAK inhibitors may be a novel preventative option in melanoma metastasis by blocking VCAM-1 expression in LNs.