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Syk inhibitors promote the expression of keratinocyte differentiation markers. (a) NHEKs were treated with Syk inhibitor, R406, for 12 and 24 hours. Then, the mRNA expression of differentiation markers were analyzed by quantitative realtime PCR. *P < 0.05 compared with DMSO-treated control group (mean AE s.e.m. n ¼ 3) (b) NHEKs were treated with R406 of different concentrations for 30 hours. Then, protein expression of differentiation markers were determined by Western blot. (c)
Source publication
Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, was initially identified as a crucial regulator in proximal immunoreceptor signaling. Additional studies have revealed its pleiotropic roles, and drugs targeting Syk are under development for inflammatory diseases. Syk expression in the skin has been detected, but its functions in the sk...
Contexts in source publication
Context 1
... used R406, an active metabolite of fosta- matinib, to treat NHEKs and detected increased mRNA expression of various differentiation markers, including involucrin, TGase 1, keratin 10, and loricrin. The induction of keratin 10 by R406 was especially prominent (Figure 2a). As shown in Figure 2b and c, R406 induced the protein expression of various differentiation markers in time-and concentration-dependent manners, and keratin 10 and lor- icrin were particularly strongly induced. ...
Context 2
... induction of keratin 10 by R406 was especially prominent (Figure 2a). As shown in Figure 2b and c, R406 induced the protein expression of various differentiation markers in time-and concentration-dependent manners, and keratin 10 and lor- icrin were particularly strongly induced. We also evaluated the effect of another Syk inhibitor, Bay 61-3606, on kerati- nocyte differentiation. ...
Context 3
... also evaluated the effect of another Syk inhibitor, Bay 61-3606, on kerati- nocyte differentiation. Similar to R406, Bay 61-3606 induced the expression of keratinocyte differentiation markers, espe- cially keratin 10 and loricrin (Figure 2d). ...
Context 4
... exam- ined if Syk also regulates PMA-and calcium-induced kerati- nocyte differentiation. As shown in Supplementary Figure S2 (online), 48 hours after knocking down Syk in NHEKs, we treated NHEKs with PMA and calcium for 24 hours. The protein expression levels of involucrin and TGase 1 induced by PMA or calcium were relatively higher in NHEKs with Syk knockdown than in controls (Supplementary Figure S2). ...
Context 5
... shown in Supplementary Figure S2 (online), 48 hours after knocking down Syk in NHEKs, we treated NHEKs with PMA and calcium for 24 hours. The protein expression levels of involucrin and TGase 1 induced by PMA or calcium were relatively higher in NHEKs with Syk knockdown than in controls (Supplementary Figure S2). ...
Citations
... SYK, also known as spleen tyrosine kinase, is involved in Th17 signaling and in keratinocyte differentiation: it stimulates Th17 cell recruitment in the skin, inducing keratinocytes to produce CCL20 [142]; regulates epidermal growth factor receptor signaling; and negatively affects keratinocyte differentiation [143]. SYK is also involved in B-cell responses, along with dendritic cell differentiation [144,145]. ...
Atopic dermatitis (AD) is the most common inflammatory skin disease, and it is considered a complex and heterogeneous condition. Different phenotypes of AD, defined according to the patient age at onset, race, and ethnic background; disease duration; and other disease characteristics, have been recently described, underlying the need for a personalized treatment approach. Recent advancements in understanding AD pathogenesis resulted in a real translational revolution and led to the exponential expansion of the therapeutic pipeline. The study of biomarkers in clinical studies of emerging treatments is helping clarify the role of each cytokine and immune pathway in AD and will allow addressing the unique immune fingerprints of each AD subset. Personalized medicine will be the ultimate goal of this targeted translational research. In this review, we discuss the changes in the concepts of both the pathogenesis of and treatment approach to AD, highlight the scientific rationale behind each targeted treatment and report the most recent clinical efficacy data.
... The epidermal growth factor receptor (EGFR) expressed in keratinocytes plays a unique role in controlling skin cell proliferation, differentiation, disorders and cancers 14 . During keratinocyte differentiation, EGFR expression and activity are downregulated 15,16 . Aberrant activation of EGFR leads to overproliferation of keratinocytes, contributing to psoriasis, while EGFR inhibitors switch keratinocytes from a proliferative to a differentiative phenotype, thus affecting epidermal development and barrier function 17 . ...
... NHEKs were used for experiments within 3 passages. For cell differentiation, we used the confluence, PMA and calcium models as we previously described 16,22 . ...
... The supernatant was removed after centrifugation, and the pellet was resuspended in DNA extraction buffer (0.2 M Na 2 HPO 4 , 0.1 M citric acid (pH 7.8)). After another centrifugation step, the pellet was stained with 80 μg/ml PI, and the cell cycle distribution was analyzed by flow cytometry (BD, FACSCalibur) as described previously 16 . ...
Decoy receptor 3 (DcR3) is a soluble receptor for Fas ligand, LIGHT and TL1A, but it also exerts effector functions. Previously, we found that DcR3 is upregulated in the serum and lesional skin of patients with psoriasis and is upregulated by EGFR activation in proliferating primary human epidermal keratinocytes. However, the functional role of intracellular DcR3 in keratinocyte differentiation is still incompletely defined. Herein, primary cultured human epidermal keratinocytes were differentiated by phorbol 12-myristate 13-acetate (PMA) treatment, calcium treatment and cell confluence, which are three standard in vitro differentiation models. We found that the constitutive expression of the DcR3 gene and protein was progressively suppressed during terminal differentiation of keratinocytes. These changes were correlated with downregulation of EGFR activation during keratinocyte differentiation. EGFR inhibition by gefitinib further decreased confluence-induced suppression of DcR3 mRNA expression, and, vice versa, knocking down DcR3 expression attenuated EGFR and EGFR ligand expression as well as EGFR activation. Under conditions without a change in cell growth, DcR3 silencing reduced the expression of involucrin and transglutaminase 1 but enhanced the induction of the terminal differentiation markers keratin 10 and loricrin. Of note, DcR3 interacted with PKCα and PKCδ and enhanced PKC activity. In keratinocytes with PKCα and PKCδ silencing, differentiation markers were differentially affected. In conclusion, DcR3 expression in keratinocytes is regulated by EGFR and forms a positive feedback loop to orchestrate constitutive EGFR and PKC activity. During differentiation, DcR3 is downregulated and involved in modulating the pattern of terminal differentiation.
... Consistently the data from the Q-PCR study indicated that PMA, TNF-α, LPS and UVB can increase Blimp-1 mRNA level ( Figure 1D). Because previously EGF was shown to upregulate Blimp-1 expression in keratinocytes (Chang et al., 2018) and EGFR can be transactivated via extracellular and intracellular manners in various cell types including keratinocytes (Nanba et al., 2013;Wu et al., 2016), we interested to explore if Blimp-1 inducers mentioned above might exert actions related to EGFR. To this end, we determined the effects of these stimuli on EGFR expression and activation. ...
... Moreover, previously we found that Syk is not only an upstream signaling molecule of EGFR in keratinocytes (Wu et al., 2016) but also can be activated by PKC in monocytes (Chang et al., 2012). Therefore, we wonder if Syk is involved in the action of PMA for Blimp-1 expression. ...
... We found that Syk activity is increased by PMA and TNF-α in HaCaT cells, and Syk inhibitor GS-9973 could suppress PMA-induced Blimp-1 increase in both HaCaT and Cal-27 cells. Because PMA can activate Syk in monocytes (Chang et al., 2012), and EGFR activation also transduces Syk signaling in keratinocytes (Wu et al., 2016) and SCC (Huang et al., 2020), we treated GS-9973 and took PMA as an example to understand the signaling cascade among PKC, Syk and EGFR. Our data that GS-9973 can reduce PMA-induced EGFR-p without affecting PKC activation suggest that PKC-Syk-EGFR-ERK-AP-1 signaling pathway is involved in Blimp-1 gene transcription. ...
B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of Blimp-1 expression and cellular functions in keratinocytes and cancer cells. Previously we demonstrated that EGF can upregulate Blimp-1 gene expression in keratinocytes, playing a negative role in regulation of cell migration and inflammation. Because it remains unclear if Blimp-1 can be regulated by other stimuli beyond EGF, here we further investigated multiple stimuli for their regulation of Blimp-1 expression in keratinocytes and squamous cell carcinoma (SCC). We found that PMA, TNF-α, LPS, polyIC, H2O2 and UVB can upregulate the protein and/or mRNA levels of Blimp-1 in HaCaT and SCC cells. Concomitant EGFR activation was observed by these stimuli, and EGFR inhibitor gefitinib and Syk inhibitor can block Blimp-1 gene expression caused by PMA. Reporter assay of Blimp-1 promoter activity further indicated the involvement of AP-1 in PMA-, TNF-α-, LPS- and EGF-elicited Blimp-1 mRNA expression. Confocal microscopic data indicated the nuclear loclization of Blimp-1, and such localization was not changed by stimuli. Moreover, Blimp-1 silencing enhanced SCC cell migration. Taken together, Blimp-1 can be transcriptionally upregulated by several stimuli in keratinocytes and SCC via EGFR transactivation and AP-1 pathway. These include growth factor PMA, cytokine TNF-α, TLR ligands (LPS and polyIC), and ROS insults (H2O2 and UVB). The function of Blimp-1 as a negative regulator of cell migration in SCC can provide a new therapeutic target in SCC.
... Normal human epidermal keratinocytes (NHEKs) were isolated from normal adult human foreskin as described previously [19]. The experiments were performed in line with the Declaration of Helsinki principles after obtaining donors' consents. ...
... Equal amounts of soluble protein from harvested cells were loaded into SDS-PAGE gel, then transferred to Immobilon-P PVDF membrane (Millipore, Billerica, MA) as described previously [19]. Different proteins were determined by specific antibodies with enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA). ...
... After dissolving in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO), absorbance was analyzed as described previously. [19] ...
UVB dosage is generally regarded as the most critical factor that determines the severity of UVB-induced skin erythema. However, recent studies have demonstrated that different UV irradiances induce varying biological responses in mouse skin even at constant UV doses. UVB-induced inflammasome activation is particularly observed in human skin keratinocytes, which are classified as immunocompetent cells, but not in mouse skin keratinocytes, which do not express sufficient inflammasome complex components. In human skin UVB-induced sunburn reactions, NLRP1 inflammasome activation critically mediates the inflammatory responses. Here, we employed primary human skin keratinocytes to explore the impact of different irradiances of a constant UVB dosage on inflammasome activation and related inflammatory responses. Our findings indicated that low-irradiance UVB induced relatively stronger NLRP1 inflammasome activation, which manifested as more active IL-1β, IL-18 release, and enhanced procaspase-1 cleavage compared to high-irradiance UVB at the same dose. Irradiance did not influence cell lysis or the expression of inflammasome complex proteins including NLRP1, proIL-1β, proIL-18, procaspase-1, and ASC. The UVB-induced TNF-α and cyclooxygenase-2 expression was also relatively higher in keratinocytes exposed to low-irradiance UVB. Low-irradiance UVB also increased reactive oxygen species production. UVB-triggered signaling analysis revealed that low-irradiance UVB resulted in more prominent p38 and JNK activation. Therefore, our findings indicated that, in addition to the role of total dosage, irradiance crucially modulates UVB-elicited inflammation in human skin keratinocytes, thus providing novel insights into human skin photobiology.
... A JAK kinázokon kívül a spleen tirozin kináz (SYK) is szerepet játszhat az AD kialakulásában (27). A SYK a dendritikus sejtek szabályozásában, a B limfociták és a keratinociták differenciációjának szabályozásában is részt vesz (28). ...
Atopic dermatitis (AD) is one of the most common inflammatory skin diseases. Despite of this, the pathomechanism of the disease is still not fully understood. In recent years, several scientific results led to the better understanding of the disease. Meantime, new therapeutical options have emerged which were developed specifically for AD patients. In this manuscript new therapies, namely dupilumab and baricitinib, which are already licenced in Hungary, are reviewed. A detailed description of therapies e.g. biological therapy, small molecular weight drugs, topical therapy and antipruritics which are currently in clinical research have been also summarized.
... To date, only a few studies have suggested a functional link between Syk and EGFR. In our previous study on human primary normal keratinocytes, we demonstrated that Syk is a downstream signal of EGFR and is involved in negatively regulating keratinocyte differentiation [14]. ...
... Protein expression was determined in cell lysates by electrophoresis and immunoblotting as previously described [14]. ...
... After observing the crucial role of Syk in skin keratinocytes [14], we further addressed the roles of Syk in skin tumour development. To this end, we first detected the expression levels of Syk and Src family kinases (SFKs) (c-Src, Lyn, Hck, and Fgr) in A431 SCC, melanoma A375, and SK-MEL-28 cells. ...
Syk is a non-receptor tyrosine kinase involved in the signalling of immunoreceptors and growth factor receptors. Previously, we reported that Syk mediates epidermal growth factor receptor (EGFR) signalling and plays a negative role in the terminal differentiation of keratinocytes. To understand whether Syk is a potential therapeutic target of cancer cells, we further elucidated the role of Syk in disease progression of squamous cell carcinoma (SCC), which is highly associated with EGFR overactivation, and determined the combined effects of Syk and PARP1 inhibitors on SCC viability. We found that pharmacological inhibition of Syk could attenuate the EGF-induced phosphorylation of EGFR, JNK, p38 MAPK, STAT1, and STAT3 in A431, CAL27 and SAS cells. In addition, EGF could induce a Syk-dependent IL-8 gene and protein expression in SCC. Confocal microscopic data demonstrated the ability of the Syk inhibitor to change the subcellular distribution patterns of EGFR after EGF treatment in A431 and SAS cells. Moreover, according to Kaplan-Meier survival curve analysis, higher Syk expression is correlated with poorer patient survival rate and prognosis. Notably, both Syk and EGFR inhibitors could induce PARP activation, and synergistic cytotoxic actions were observed in SCC cells upon the combined treatment of the PARP1 inhibitor olaparib with Syk or the EGFR inhibitor. Collectively, we reported Syk as an important signalling molecule downstream of EGFR that plays crucial roles in SCC development. Combining Syk and PARP inhibition may represent an alternative therapeutic strategy for treating SCC.
... Using the meta-analysis derived atopic dermatitis transcriptome (MADAD) [26] as a reference core AD transcriptome, Pavel et al. presented data which supported the usage of dual JAK/SYK inhibition in AD by showing a dose-dependent improvement of the transcriptomic profile. The rational of this approach refers to the inhibition of T cell polarization by JAK inhibition [239] as well as improvement of the terminal epidermal differentiation by SYK inhibitors [240]. Additionally, IL31 expression was reduced more efficiently by the dual inhibition approach than that seen in dupilumab treatment. ...
During the last decades, high-throughput assessment of gene expression in patient tissues using microarray technology or RNA-Seq took center stage in clinical research. Insights into the diversity and frequency of transcripts in healthy and diseased conditions provide valuable information on the cellular status in the respective tissues. Growing with the technique, the bioinformatic analysis toolkit reveals biologically relevant pathways which assist in understanding basic pathophysiological mechanisms. Conventional classification systems of inflammatory skin diseases rely on descriptive assessments by pathologists. In contrast to this, molecular profiling may uncover previously unknown disease classifying features. Thereby, treatments and prognostics of patients may be improved. Furthermore, disease models in basic research in comparison to the human disease can be directly validated. The aim of this article is not only to provide the reader with information on the opportunities of these techniques, but to outline potential pitfalls and technical limitations as well. Major published findings are briefly discussed to provide a broad overview on the current findings in transcriptomics in inflammatory skin diseases.
... 91 Spleen Tyrosine Kinase is a nonreceptor tyrosine kinase (Fig 2), and the SYK pathway is involved in several cytokine signaling immune pathways, with subsequent regulation on Th17, B-cell, and dendritic cells activation, as well as inhibition on keratinocyte terminal differentiation. [92][93][94][95][96][97][98][99][100][101] Concomitant JAK and SYK inhibition had synergistic, beneficial anti-inflammatory effects, 97,102 providing the rationale to add SYK inhibition to JAK antagonism as a potential added therapeutic value to AD. ...
Objective:
Atopic dermatitis (AD) is an increasingly common inflammatory skin disease undergoing significant revolution in recent years. New data on disease pathogenesis advanced the developments of novel therapeutics, mainly for patients with moderate to severe conditions, for whom treatment options have been largely insufficient for many years.
Data sources:
Review of recent studies investigating systemic treatments for AD.
Study selections:
Relevant literature concerning novel therapeutics for AD beyond targeted monoclonal antibodies antagonizing selectively interleukin (IL)-4 or IL-13 was obtained from a PubMed and clinicaltrials.gov search and summarized.
Results:
Multiple clinical trials of both nonspecific as well as specific agents revealed favorable outcomes in AD, including JAK inhibitors, a dual JAK/SYK inhibitor, a histamine H4R antagonist, antagonists of the TSLP/OX40L axis, an IL-22 inhibitor, and IL-33 and IL-17C antagonists. Importantly, negative trials were published as well (eg, phosphodiesterase 4 inhibitor, apremilast).
Conclusion:
In this rapidly evolving field of AD treatments, a completely new treatment paradigm will be available in the near future.
... Spleen tyrosine kinases (SYKs) are involved in the release of cytokines during the proinflammatory process, including IL-1b, IL-10 and IL-17, and regulate DCs, B lymphocytes and keratinocyte differentiation, suggesting that SYK inhibitors could improve inflammatory skin diseases with aberrant differentiation, such as AD [78,79]. ASN002 is a potent, dual inhibitor of JAK and SYK kinases [47]. ...
Atopic dermatitis (AD) is the most common inflammatory skin disease driven by both terminal keratinocyte differentiation defects and type 2 immune responses, and this condition causes psychological and social morbidity. Although patients with severe AD require systemic immunotherapy, conventional agents including ciclosporin could not be used for several years due to side effects such as nephrotoxicity, hypertension and long-term risks of malignancy. It is well known that dupilumab, which blocks receptor binding of both IL-4 and IL-13, is remarkably efficacious in the treatment of AD. We have entered a new era when many novel topical and systemic agents that may have great potential in AD treatment are emerging through clinical trials. The purpose of this article is to summarize the efficacy and safety of the current topical and systemic therapies in AD by reviewing recently published papers regarding phase II/III clinical trials. It is revealed that topical phosphodiesterase 4 inhibitors and Janus kinase (JAK) inhibitors are promising treatments for AD. Moreover, systemic therapies such as biologics targeting IL-13 and oral JAK inhibitors show strong efficacy in AD.
... [26][27][28]31,32 Spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, regulates B-cell and dendritic cell (DC) differentiation and T H 17/IL-17 signaling and inhibits keratinocyte terminal differentiation. [33][34][35][36][37][38][39][40][41] In mice SYK inhibition was shown to inhibit T H 17 pathway signaling and improve terminal epidermal differentiation. [41][42][43] Thus dual JAK-SYK inhibition might broaden the range of cytokines targeted to successfully resolve AD across the different disease subtypes. ...
... [33][34][35][36][37][38][39][40][41] In mice SYK inhibition was shown to inhibit T H 17 pathway signaling and improve terminal epidermal differentiation. [41][42][43] Thus dual JAK-SYK inhibition might broaden the range of cytokines targeted to successfully resolve AD across the different disease subtypes. 9,10,12,21,[44][45][46][47][48] ASN002 is the first oral dual JAK/SYK inhibitor tested in patients with AD. ...
... No significant changes were seen in the placebo group at both day 15 and day 29, and the 20-mg group showed significant but modest changes from [34][35][36][37][38][39] and inhibits keratinocyte terminal differentiation. [34][35][36][37][38][39][40][41]83 The rapid inhibition of various axes and amelioration of barrier pathology can be induced by the broadened spectrum of cytokines targeted with JAK/SYK inhibition. 31,[35][36][37][38]84,85 Previous studies have shown that concomitant JAK/SYK inhibition has a synergistic therapeutic effect on inflammatory diseases. ...
Background:
Moderate-to-severe atopic dermatitis (AD) has been associated with significant disease burden and systemic abnormalities and often requires systemic treatments. Currently, safe and effective oral systemic treatments for moderate-to-severe AD are not yet available. ASN002 is an oral inhibitor of the Janus kinase/spleen tyrosine kinase signaling pathways, targeting several cytokine axes (TH2/TH22/TH17/TH1) and epidermal differentiation.
Objective:
We sought to evaluate the effect of ASN002 on the cellular and molecular biomarker profile of patients with moderate-to-severe AD and to correlate changes in biomarkers to improvements in clinical severity measures and pruritus.
Methods:
Thirty-six patients with moderate-to-severe AD were randomized to groups with dose escalation of ASN002 (20, 40, and 80 mg) and a placebo group. Skin biopsy specimens were performed at baseline, day 15, and day 29. Gene expression studies were conducted by using microarray and quantitative RT-PCR, and cellular infiltrates and protein expression were studied by using immunohistochemistry.
Results:
ASN002 reversed the lesional skin transcriptome toward a nonlesional phenotype. It also rapidly and significantly suppressed key inflammatory pathways implicated in AD pathogenesis, including TH2 (IL4 receptor [IL4R], IL13, CCL13/monocyte chemoattractant protein 4, CCL17/thymus and activation-regulated chemokine, CCL18/pulmonary and activation-regulated chemokine, CCL22/macrophage-derived chemokine, and CCL26/eotaxin-3), TH17/TH22 (lipocalins, PI3/elafin, CCL20, S100A7/S100A8/S100A9, and IL36G/IL36RN), and TH1 (IFNG, CXCL9/CXCL11, and MX1) axes and barrier-related measures (filaggrin [FLG] and CLDN23). Significant improvements in AD gene signatures were observed predominantly in the 40- and 80-mg groups. Smaller and largely nonsignificant molecular changes were seen in the 20-mg and placebo groups.
Conclusion:
The Janus kinase/spleen tyrosine kinase inhibitor ASN002 significantly suppressed key AD inflammatory pathways, corresponding to clinical response. ASN002 might be an effective novel therapeutic agent for moderate-to-severe AD.
