Versatile Role of Prokineticins and Prokineticin Receptors in Neuroinflammation

Prokineticins are a new class of chemokine-like peptides involved in a wide range of biological and pathological activities. In particular, prokineticin 2 (PK2), prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) play a central role in modulating neuroinflammatory processes. PK2 and PKRs, which are physiologically expressed at very low levels, are strongly upregulated during inflammation and regulate neuronal-glial interaction. PKR2 is mainly overexpressed in neurons, whereas PKR1 and PK2 are mainly overexpressed in astrocytes. Once PK2 is released in inflamed tissue, it is involved in both innate and adaptive responses: it triggers macrophage recruitment, production of pro-inflammatory cytokines, and reduction of anti-inflammatory cytokines. Moreover, it modulates the function of T cells through the activation of PKR1 and directs them towards a pro-inflammatory Th1 phenotype. Since the prokineticin system appears to be upregulated following a series of pathological insults leading to neuroinflammation, we will focus here on the involvement of PK2 and PKRs in those pathologies that have a strong underlying inflammatory component, such as: inflammatory and neuropathic pain, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, stroke, obesity, diabetes, and gastrointestinal inflammation.

Serum Prokineticin-2 in Prepubertal and Adult Klinefelter Individuals

The prokineticin-2 (PROK2) is a small peptide belonging to the prokineticin family. In humans and rodents this chemokine is primarily involved in the control of central and peripheral reproductive processes. Klinefelter's syndrome (KS) is the first cause of male genetic infertility, due to an extra X chromosome, which may occur with a classical karyotype (47, XXY) or mosaic forms (46, XY/47, XXY). In affected subjects, pubertal maturation usually begins at an adequate chronological age, but when development is almost complete, they display a primary gonadal failure, with early spermatogenesis damage, and later onset of testosterone insufficiency. Thus, the main aim of the present study was to investigate the serum levels of PROK2 in prepubertal and adult KS patients, comparing them with healthy subjects. We showed for the first time the presence of PROK2 in the children serum but with significant changes in KS individuals. Indeed, compared to healthy subjects characterized by PROK2 serum elevation during the growth, KS individuals showed constant serum levels during the sexual maturation phase (higher during the prepubertal phase but lower during the adult age). In conclusion, these data indicate that in KS individuals PROK2 may be considered a biomarker for investigating the SK infertility process.

Differential inflammation-mediated function of prokineticin 2 in the synovial fibroblasts of patients with rheumatoid arthritis compared with osteoarthritis

Prokineticin 2 (PK2) is a secreted protein involved in several pathological and physiological processes, including the regulation of inflammation, sickness behaviors, and circadian rhythms. Recently, it was reported that PK2 is associated with the pathogenesis of collagen-induced arthritis in mice. However, the role of PK2 in the pathogenesis of rheumatoid arthritis (RA) or osteoarthritis (OA) remains unknown. In this study, we collected synovial tissue, plasma, synovial fluid, and synovial fibroblasts (SF) from RA and OA patients to analyze the function of PK2 using immunohistochemistry, enzyme-linked immunosorbent assays, and tissue superfusion studies. PK2 and its receptors prokineticin receptor (PKR) 1 and 2 were expressed in RA and OA synovial tissues. PKR1 expression was downregulated in RA synovial tissue compared with OA synovial tissue. The PK2 concentration was higher in RA synovial fluid than in OA synovial fluid but similar between RA and OA plasma. PK2 suppressed the production of IL-6 from TNFα-prestimulated OA-SF, and this effect was attenuated in TNFα-prestimulated RA-SF. This phenomenon was accompanied by the upregulation of PKR1 in OA-SF. This study provides a new model to explain some aspects underlying the chronicity of inflammation in RA.

Oligogenic Inheritance Underlying Incomplete Penetrance of PROKR2 Mutations in Hypogonadotropic Hypogonadism

The role of the prokineticin 2 pathway in human reproduction, olfactory bulb morphogenesis, and gonadotropin-releasing hormone secretion is well established. Recent studies have highlighted the implication of di/oligogenic inheritance in this disorder. In the present study, we aimed to identify the genetic mechanisms that could explain incomplete penetrance in hypogonadotropic hypogonadism (HH). This study involved two unrelated Tunisian patients with HH, which was triggered by identifying a homozygous p.(Pro290Ser) mutation in the PROKR2 gene in a girl (HH1) with Kallmann syndrome (KS). The functional effect of this variant has previously been well demonstrated. Unexpectedly, her unaffected father (HH1P) and brother (HH1F) also carried this genetic variation at a homozygous state. In the second family, we identified a heterozygous p.(Lys205del) mutation in PROKR2, both in a male patient with normosmic idiopathic IHH (HH12) and his asymptomatic mother. Whole-exome sequencing in the three HH1 family members allowed the identification of additional variants in the prioritized genes. We then carried out digenic combination predictions using the oligogenic resource for variant analysis (ORVAL) software. For HH1, we found the highest number of disease-causing variant pairs. Notably, a CCDC141 variant (c.2803C > T) was involved in 18 pathogenic digenic combinations. The CCDC141 variant acts in an autosomal recessive inheritance mode, based on the digenic effect prediction data. For the second patient (HH12), prediction by ORVAL allowed the identification of an interesting pathogenic digenic combination between DUSP6 and SEMA7A genes, predicted as “dual molecular diagnosis.” The SEMA7A variant p.(Glu436Lys) is novel and predicted as a VUS by Varsome. Sanger validation revealed the absence of this variant in the healthy mother. We hypothesize that disease expression in HH12 could be induced by the digenic transmission of the SEMA7A and DUSP6 variants or a monogenic inheritance involving only the SEMA7A VUS if further functional assays allow its reclassification into pathogenic. Our findings confirm that homozygous loss-of-function genetic variations are insufficient to cause KS, and that oligogenism is most likely the main transmission mode involved in Congenital Hypogonadotropic Hypogonadism.

Prokineticin-2 prevents neuronal cell deaths in a model of traumatic brain injury

Prokineticin-2 (Prok2) is an important secreted protein likely involved in the pathogenesis of several acute and chronic neurological diseases through currently unidentified regulatory mechanisms. The initial mechanical injury of neurons by traumatic brain injury triggers multiple secondary responses including various cell death programs. One of these is ferroptosis, which is associated with dysregulation of iron and thiols and culminates in fatal lipid peroxidation. Here, we explore the regulatory role of Prok2 in neuronal ferroptosis in vitro and in vivo. We show that Prok2 prevents neuronal cell death by suppressing the biosynthesis of lipid peroxidation substrates, arachidonic acid-phospholipids, via accelerated F-box only protein 10 (Fbxo10)-driven ubiquitination, degradation of long-chain-fatty-acid-CoA ligase 4 (Acsl4), and inhibition of lipid peroxidation. Mice injected with adeno-associated virus-Prok2 before controlled cortical impact injury show reduced neuronal degeneration and improved motor and cognitive functions, which could be inhibited by Fbxo10 knockdown. Our study shows that Prok2 mediates neuronal cell deaths in traumatic brain injury via ferroptosis.

Pressure Overload–Mediated Sustained PKR2 (Prokineticin-2 Receptor) Signaling in Cardiomyocytes Contributes to Cardiac Hypertrophy and Endotheliopathies

Chronic cardiac pressure overload, caused by conditions, such as hypertension, induces pathological hypertrophic growth of myocardium and vascular rarefaction, with largely unknown mechanisms. Here, we described that expression of the PKR2 (prokineticin-2 receptor) is increased in the cardiomyocytes of mice following transaortic constriction pressure overload–mediated pathological hypertrophy. To identify PKR2-induced pathways, we performed microarray analysis on TG-PKR2 (transgenic mice overexpressing cardiomyocyte-restricted human PKR2) hearts and cytokine analyses in hPKR2 overexpressing H9c2-lines (PKR2-cardiomyocytes). An enrichment of activin pathway gene sets was found in both TG-PKR2 and transaortic constriction-operated hearts. Elevated levels of 2 cytokines activin A and its coreceptor, sENG (soluble Endoglin), were found in both PKR2-cardiomyocytes and in PKR2-cardiomyocytes conditioned medium. ELISA analyses of the cardiomyocytes derived from both TG-PKR2 and transaortic constriction hearts revealed high levels of these cardiokines that were repressed with antibodies blocking PKR2, indicating a PKR2-dependent event. The conditioned medium of PKR2-cardiomyocytes induced fenestration of endothelial cells and inhibited tube-like formations. These endotheliopathies were blocked by either depleting activin A or sENG from conditioned medium or by using 2 pharmacological inhibitors, follistatin, and TRC105. In addition, similar endotheliopathies were produced by exogenous administration of activin A and ENG. Prolonged exposure to prokineticin-2 in PKR2-cardiomyocytes increased cell volume by the PKR2/Gα 12/13 /ERK5-pathway. Activation of the PKR2/Gα 12/13 /matrix metalloprotease-pathway promoted both activin A and sENG release. This study reveals that pressure overload–mediated PKR2 signaling in cardiomyocytes contributes to cardiac hypertrophy through autocrine signaling, and vascular rarefaction via cardiac cytokine-mediated cardiomyocyte–endothelial cell communications. Our results may contribute to the development of potential therapeutic targets for heart failure.

Circulating Prokineticin 2 Levels Are Increased in Children with Obesity and Correlated with Insulin Resistance

Objective. Prokineticin 2 (PK2) has been shown to regulate food intake, fat production, and the inflammation process, which play vital roles in the pathogenesis of obesity. The first aim of this study was to investigate serum PK2 levels in children with obesity and normal-weight children. The second aim was to compare the levels of PK2 between children with obesity, with and without nonalcoholic fatty liver disease (NAFLD). Methods. Seventy normal-weight children and 91 children with obesity (22 with NAFLD) were recruited. Circulating PK2, IL-6, and TNF-α were measured by enzyme-linked immunosorbent assays. Anthropometric and biochemical measurements related to adiposity, lipid profile, and insulin resistance were examined for all participants. Results. Serum PK2 was significantly higher in children with obesity than in the normal-weight controls. Circulating PK2 levels were not different between the patients with and without NAFLD. Circulating PK2 was positively correlated with BMI, BMI z-score, insulin, glucose, HOMA-IR, total cholesterol, low-density lipoprotein cholesterol, alanine aminotransferase, and gamma-glutamyl transpeptidase. Binary logistic regression revealed that the odds ratios for obesity were significantly elevated with increasing PK2. Conclusions. PK2 was strongly associated with obesity, and it may also be related to metabolic disorders and insulin resistance. This trial is registered with ChiCTR2000038838.

Differential inflammation-mediated function of prokineticin 2 in the synovial fibroblasts of patients with rheumatoid arthritis compared to osteoarthritis

Prokineticin 2 (PK2) is a secreted protein involved in several pathological and physiological processes, including the regulation of inflammation, sickness behaviors, and the circadian rhythm. Recently, it was reported that PK2 is associated with the pathogenesis of collagen-induced arthritis in mice. However, whether PK2 influences the pathogenesis of rheumatoid arthritis (RA) or osteoarthritis (OA) remains unknown. In this study, we collected synovial tissue, plasma, synovial fluid, and fibroblast-like synoviocytes (FLS) from RA and OA patients to analyze the role of PK2 using immunohistochemistry, ELISAs, and tissue superfusion studies. PK2 and its receptors prokineticin receptor (PKR) 1 and 2 were expressed in RA and OA synovial tissues. PKR1 expression in RA synovial tissue was downregulated compared with OA synovial tissue. The PK2 concentration was higher in RA synovial fluid than in OA synovial fluid but similar between RA and OA plasma. PK2 suppressed the production of IL-6 from TNFα-prestimulated OA-FLS, and this effect was attenuated in TNFα-prestimulated RA-FLS. This phenomenon was accompanied by the upregulation of PKR1 in OA-FLS and the downregulation of PK2 and PKR1 in RA-FLS. This study provides a new model to explain some aspects underlying the chronicity of inflammation in RA.