The Emerging Role of Never-in-Mitosis A - Related Kinases in the Endothelium

Abstract

The endothelium forms a monolayer, which functions to ensure tissue homeostasis. Barrier hyperpermeability has been associated with lung, brain and eye disease. An emerging body of evidence reports the involvement of Never-in-Mitosis A - Related Kinases in vascular responses, suggesting their potential value as potential therapeutic targets in endothelial-dependent disorders.

Barrier Function and Never-in-Mitosis A-Related Kinases:

The endothelial permeability is affected by a plethora of cellular and environmental stimuli, and it is regulated by a meticulously organized molecular network, to safeguard homeostasis under a diverse variety of stresses. Several kinases have been identified to mediate–and potentiate-inflammatory responses, which in turn induce tissue dysfunction. An emerging body of evidence supports the involvement of Never-in-Mitosis A (NIMA)-Related Kinases - or NEKs - in endothelium-related disorders.

The first NIMA-related gene was discovered in Aspergillus nidulans. Studies in yeasts (e.g. Saccharomyces cerevisiae), unicellular (e.g. Chlamydomonas) and multicellular (e.g. Drosophila, Xenopus) organisms demonstrated the essential role of NEKs in cytokinesis. Humans express 11 serine/threonine NEKs which have been involved in disease pathogenesis; including ciliopathy, diabetes, brain dysfunction and cancers (Table I).

Table I:

The Role of Never-in-Mitosis A - Related Kinases (NEKs) in Cell Function and Dysfunction

NEKs	Description	Function in the cells	NEK-related cell disorders and disease	References
NEK1	• Primarily localized in the cytosol, nucleus and mitochondria • Expressed in germ cells • M.W. ≈125 kDa	• Centrosome duplication and microtubule organization during cell division • Cell cycle progression • Genomic stability • Involved in meiosis and neural development	• Polycystic kidney disease (PKD) • Retinal degeneration • Neurological abnormalities • Impaired DNA damage repair mechanisms	[1–2]
NEK2	• Localized to centrosomes, spindle pole and kinetochores • Expressed in female germs, HeLa and KE37 cancer cells • M.W. ≈46 kDa	• Controls centrosome separation and bipolar spindle formation in mitotic cells • Involved in chromatin condensation in meiotic cells • Promotes centrosome splitting • Ensures accurate chromosome segregation during G2/M phase • Regulation of endothelial permeability	• Dysregulation of centrosome cycle and barrier function • Aneuploid • Aberrant cell cycle progression and genomic instability • Lung Metastasis • Drug resistance promotion	[4, 6–10]
NEK3	• Located in the cytoplasm, axons and post-mitotic neurons • Highly expressed in the brain • M.W. ≈50 kDa	• Involved in cell cycle progression and proliferation • Promotes cell migration and invasion • Controls microtubule dynamics	• NEK3 overexpression is detected in thyroid cancer • Mutations disrupt microtubule deacetylation and polarity • Dysregulated apoptosis and DNA damage response	[25]
NEK4	• Localized to nuclear speckles, cilia root and PML bodies • Expressed in HEK293T, heart and breast cancer cells. • • M.W. ≈46 kDa	• Involved in cell cycle arrest in response to double-stranded DNA damage • Maintains cilium integrity • Required for normal entry replicative senescence	• Ciliopathies. • Microtubule poisoning • Associated with diabetic nephropathy • Lung cancer promotion	[11–12]
NEK5	• Predominantly localized in the cytoplasm and nucleus • 708-amino acid protein. • M.W. ≈59 kDa	• Regulates centrosome separation and mitotic spindle assembly • Promotes cancer cell proliferation via Cyclin A2 • Regulates cysteine-type endopeptidase activity and muscle cell differentiation	• Overexpression of NEK5 enhances rough acinar morphology • Cancer progression • Promotion of breast epithelial cancers	[21, 25]
NEK6	• Localized to the cytoplasm and in centrosomes • Highest expression in the heart and skeletal muscle • M.W. ≈45 kDa	• Regulates redox balance and DNA damage response. • Required for metaphase progression • Involved in cancer cell transformation	• Errors in chromosome segregation, cytokinesis, and aberrant mitotic progression • Genomic instability • Alters apoptosis • Suppresses p53-induced cancer cell senescence	[10, 13–15]
NEK7	• Smallest NEK kinase in mammals • Localized to the cytoplasm • Expressed in lung, liver, spleen, testis, muscle, heart, leukocyte and brain • M.W. ≈32 kDa	• Essential for mitotic spindle formation and cytokinesis • Regulates microtubule stability and nucleation	• Induced in breast, hepatocellular, colorectal and lung cancer. • Formation of pores in the cell membrane • Increase in the number of mitotic cells acquiring a multipolar or monopolar spindle phenotype, which ultimately leads to cell arrest • Associated with Type 2 diabetes, blood brain barrier dysfunction	[13, 16–17]
NEK8	• Primarily localized in centrosomes. • Expressed to the cilia, small hair-like structures on the surface of cells • M.W. ≈66 kDa	• Involved in cell cycle progression from G2 to M phase • Regulates cytoskeletal structure in kidney tubule epithelial cells • Associated with ciliogenesis, cell cycle progression, and DNA damage response	• NEK8 mutations lead to Nephronophthisis. • Juvenile autosomal recessive form of polycystic kidney disease • Associated with ciliopathy and polycystic kidney disease	[16, 21, 25]
NEK9	• Located on chromosome 14q2, cytoplasm and nucleus • 979 - amino acid protein. • Highly expressed in liver, heart, kidney and testis • M.W. ≈100 kDa	• NEK9 knockdown induces G1 arrest • Involved in centrosome maturation, duplication and microtubule organization • Modulation of spindle organization • Involved in vascular permeability	• NEK9 mutation causes a lethal skeletal dysplasia • NEK9 depletion inhibits cell growth in p53-mutant cancer cells • Overexpression enhances tumor invasion in non-small cell lung cancer • NEK9 suppression induces endothelial dysfunction	[6, 18–20]
NEK10	• Localized to chromosome 3 • 712 - amino acid protein • Mainly expressed in the brain • M.W. ≈50 kDa	• Involved in cellular response to UV irradiation • Mediates G2/M cell cycle arrest, MEK and ERK1/2 activation • Participates in genotoxic stress response and DNA replication	• NEK10 promotes lung cancer metastasis • NEK10 mutation causes bronchiectasis • Associated with primary ciliary dyskinesia	[21–23]
NEK11	• Localized to the nucleoli • Expressed in trachea, lung, uterus, appendix and cerebellum • M.W. ≈70 kDa	• Involved in DNA replication, genotoxic stress response, apoptosis and S phase checkpoint transition	• Loss of NEK11 prevents G2/M arrest • NEK11 promotes cell death in colorectal cancer cells • Associated with short-rib thoracic dysplasia 6 and melanoma.	[19, 24]

NEK1 in the blood brain barrier:

NEK1 is involved in cell cycle, cilia formation, DNA-damage response, microtubule stability, and neural development regulation. Excess NEK1 catalytic activity leads to primary glia loss. NEK1 deficient cells display defective G1/S and G2/M checkpoints and impaired DNA repair after ionizing radiation [1]. NEK1 - deficient mice demonstrated postnatal lethality due to cerebrovascular endothelial cell necroptosis [2].

NEK2 involvement in P53 regulation and endothelial permeability:

NEK2 is closely related to the prototypic Aspergillus nidulans NIMA. It promotes the splitting of duplicated centrosomes via centriolar protein phosphorylation [3]. NEK2-mediated P53 phosphorylation at Ser 315 reduces P53 stability [4]. This tumor suppressor regulates the Ras-related C3 botulinum toxin substrate 1/ Ras homolog family member A balance in the endothelium and supports vascular integrity (Figure 1) [5].

FIGURE 1:

Never in Mitosis A Related Kinases (NEKs) in endothelial function and dysfunction: A) Cell injury, reactive oxygen species (ROS) generation and inflammation induces endothelial hyperpermeability. This is a key feature of acute respiratory distress syndrome (ARDS), sepsis, keratitis, Fuch’s dystrophy, thyroid eye disease, blood brain barrier dysfunction; which also contributes in cancer metastasis. B) NEK2, NEK3, NEK4, NEK7, NEK9 and NEK10 are overexpressed in several disorders, including sepsis and cancer. NEK inhibition, by suppressing inflammation, abnormal growth and barrier dysfunction, can alleviate endothelium-related disease. C) Growth hormone-releasing hormone (GHRH) antagonists and heat shock protein 90 (Hsp90) inhibitors activate the unfolded protein response, which in turns induces P53. This is a tumor suppressor protein associated with barrier enhancement activities. P53 balances the opposing activities of Ras-related C3 botulinum toxin substrate 1 and Ras homolog gene family member A in the endothelium. Moreover, P53 phosphorylates cofilin (pCofilin) to suppress its actin-severing activity. D) Inflammatory bacterial toxins (e.g. liposaccharides, lipoteichoic acid) can modify P53 activity and stability via phosphorylation. At least three NEK family members, namely NEK2, NEK9 and NEK10, phosphorylate P53 (pP53). That event leads to P53 degradation, myosin light chain 2 phosphorylation (pMLC2), filamentous (F) actin formation and endothelial leak.

Bovine pulmonary artery endothelial cells (BPAEC) expressing less NEK2 due to NCL00017509 treatment or siRNA transfection presented with compromised barrier function. NCL00017509 is a potent and reversible NEK2 inhibitor. Moreover, those cells were more vulnerable to the Gram-negative bacterial endotoxin Lipopolysaccharides (LPS), as compared to the corresponding control groups [6]. Furthermore, NEK2 was induced in mouse lungs subjected to LPS treatment, or cecal ligation and puncture operation [6] (Figure 1). Growth hormone - releasing hormone antagonists, which activate the unfolded protein response, suppress NEK2 induction due to LPS and IFN-gamma in endothelial cells [7]. Lung analysis of non-small cell lung carcinoma patients revealed that high levels of NEK2 expression is associated with tumor stage and progression [8] and promotes drug resistance [9]. Moreover, elevated expression levels of NEK2 mRNA is associated with poor prognosis in lung cancer patients [8].

NEK4 in endothelial hypoxia:

NEK4 is involved in post-mitotic cell cilia, regulates the entry into replicative senescence, and participates in DNA damage responses. Gene expression analysis in human pulmonary artery endothelial cells revealed that hypoxic conditions affect NEK4 levels [10]. NEK4 overexpression resulted to decreased levels of VE-Cadherin, and promoted lung cancer metastasis via epithelial-to-mesenchymal transition interference. Furthermore, NEK-4 knockdown reduced tumor formation and metastasis [11].

NEK6 in lung cancer:

NEK6 and NEK7 have 85% sequence identity [12]. NEK6 expression levels were increased in human non-small lung carcinoma lung tissues, as compared to paired non-tumor tissues, and were associated to metastatic progression [13]. NCI-H1299 and NCI-H1975 lung cancer cells express high levels NEK6, and specific inhibitors are being developed - and tested - against cancers [14]. NEK6 mRNA levels were associated with poor prognosis in pulmonary malignancies [13].

NEK7 in retinopathy and brain injury:

NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux [15]. Retinal endothelial cell NEK7 inhibition ameliorates high glucose induced-human endothelial cell dysfunction, a complication of diabetic retinopathy [15]. Progressive endothelial eye disorders may lead to blindness. In Fuchs’ endothelial corneal dystrophy, corneal transplantation is the only therapeutic option; and the majority of Graves’ disease patients present with thyroid eye ophthalmopathy [16–19].

Blood brain barrier (BBB) endothelial dysfunction is associated with traumatic brain injury, neurodegenerative disorders and subarachnoid hemorrhage (SAH). NEK7 expression was increased in experimental SAH. Moreover, the induction of that kinase aggravated neurological deficits, brain edema, and BBB permeability. On the other hand, NEK7 small interfering RNA injection reversed those effects [20]. The aforementioned kinase was induced in the lungs of septic mice.

NEK9 in lung inflammatory disease and permeability:

NEK9 activation phosphorylates NEK6 and NEK7 to modulate kinesin-like protein 11 and microtubule formation, and to ensure proper cytoskeletal and mitotic spindle arrangement [21]. In lung cancers, co-expression of NEK9 and mutant p53 promoted cell proliferation via mitogen activated protein kinase 14 upregulation [22]. LPS induced NEK9 expression in endothelial cells and in septic mice. Targeted suppression of that kinase alleviated LPS-induced barrier dysfunction [6]. It was suggested that myosin light chain phosphorylation – which increases endothelial permeability - is regulated by NEK9, suggesting the development of new pharmacotherapies based on NEK9 modulation [23].

NEK10 and NEK11 in stress responses:

NEK10 controls the G2/M checkpoint and phosphorylates P53 on tyrosine 327, to modulate p53-responsive genes in the context of growth, DNA replication, and stress response [24] (Table I). This is important because P53 is a key regulator of barrier integrity, and mediates - at least in part - the protective activities of GHRH antagonists and heat shock protein 90 inhibitors in the endothelium [25]. NEK10 regulates β-catenin turnover. A549 cells deficient for that kinase exerted compromised ability to form tumors in vitro, and could not metastasize to mouse lung tissues after tail vein injection [26]. NEK11, which is phosphorylated by NEK2, is involved in DNA replication and genotoxic stress responses [27]. Increased mRNA levels of NEK11 were associated with poor prognosis in non - small - cell lung carcinoma (Table I)[28].

Enzyme inhibitors in clinics:

Enzyme-based therapeutics have been successfully applied in clinical trials. The β-Hydroxy β-methylglutaryl-CoA reductase inhibitors reduce ameliorate cardiovascular disease. Angiotensin-converting enzyme inhibitors manage hypertension and reduce mortality following myocardial infarction. Viral protease inhibitors are used to treat HIV infections. Hence, the premise. Elevated NEK expression levels have been associated with human disease (Table I), and at least 2 NEKs modulate barrier function [6]. Hence, the development of NEK inhibitors [29] appears to be an appealing strategy to treat endothelial-related disorders (e.g. lung injury, sepsis, keratitis) (Figure 1) [29].

Future Directions:

Many questions on the role of NEKs in endothelial function remain unanswered. Which is the exact role of those proteins in cytoskeletal protein modulation (e.g. VE-Cadherin, zonula occludens); transcellular/paracellular permeability; and inflammatory response in response to bacterial infection or injury? The potential interrelations of NEKs and unfolded protein response (UPR) in the endothelial context would be an intriguing topic of research. There is a positive regulation of P53 and UPR in lung endothelial cells, and P53 serves as a NEK substrate (Figure 1). Moreover, at least one UPR sensor, namely the activating transcription factor 6 (ATF6), protects against endothelial barrier dysfunction, renal/cerebral ischemia/reperfusion, and myocardial infarction [30].

Conclusive remarks:

The flawless transport of nutrients and gases through the endothelial semipermeable monolayers is crucial for tissue function and survival. Endothelial hyperpermeability is associated with acute lung injury, acute respiratory distress syndrome (ARDS) related or not to COVID-19, keratitis, BBB dysfunction, thyroid ophthalmopathy (Table I). In our view, the potential beneficial effects of NEK inhibitors in experimental models of endothelial-related illness should be explored, since there is an emerging body of evidence suggesting that those compounds can block vascular leak. The devastating outcomes of the ARDS-related mortality of the recent pandemic (>7 million deaths worldwide) suggest that the aforementioned investigations should be considered to be of the highest priority.

Key Facts:

• Endothelial dysfunction has been associated with severe disease, including acute respiratory distress syndrome.

• NEKs are involved in cell motility and division.

• An emerging body of evidence supports the involvement of NEKs in barrier regulation.

• NEK inhibitors may represent a new therapeutic possibility in disorder related to endothelial barrier function.