February 2017
Volume 58, Issue 2
Open Access
Biochemistry and Molecular Biology  |   February 2017
Activation of p38 and Erk Mitogen-Activated Protein Kinases Signaling in Ocular Rosacea
Author Affiliations & Notes
  • Edward J. Wladis
    Department of Ophthalmology, Albany Medical College, Albany, New York, United States
    Department of Ophthalmology, Ophthalmic Plastic Surgery, Lions Eye Institute, Slingerlands, New York, United States
  • Supraja Swamy
    Medical Education Program, Albany Medical College, Albany, New York, United States
  • Alyssa Herrmann
    Medical Education Program, Albany Medical College, Albany, New York, United States
  • Jinhong Yang
    Medical Education Program, Albany Medical College, Albany, New York, United States
  • J. Andrew Carlson
    Department of Pathology, Albany Medical College, Albany, New York, United States
  • Alejandro P. Adam
    Department of Ophthalmology, Albany Medical College, Albany, New York, United States
    Department of Molecular and Cellular Physiology, Albany Medical College, Albany, New York, United States
  • Correspondence: Alejandro P. Adam, Department of Molecular and Cellular Physiology, Albany Medical College (ME-600B1 MC-8), 43 New Scotland Avenue, Albany, NY 12208, USA; adama1@mail.amc.edu
Investigative Ophthalmology & Visual Science February 2017, Vol.58, 843-848. doi:10.1167/iovs.16-20275
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Edward J. Wladis, Supraja Swamy, Alyssa Herrmann, Jinhong Yang, J. Andrew Carlson, Alejandro P. Adam; Activation of p38 and Erk Mitogen-Activated Protein Kinases Signaling in Ocular Rosacea. Invest. Ophthalmol. Vis. Sci. 2017;58(2):843-848. doi: 10.1167/iovs.16-20275.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: Rosacea-related cutaneous inflammation is a common cause of ocular surface disease. Currently, there are no specific pharmacologic therapies to treat ocular rosacea. Here, we aimed at determining the differences in intracellular signaling activity in eyelid skin from patients with and without ocular rosacea.

Methods: This was an observational, comparative case series including 21 patients undergoing lower lid ectropion surgery at one practice during 2013 and 2014 (18 patients with rosacea, 13 control patients), and 24 paraffin-embedded archival samples from Albany Medical Center, selected randomly (12 patients with rosacea, 12 control patients). Cutaneous biopsies resulting from elective lower lid ectropion surgery were analyzed by Proteome Profiler Human Phospho-Kinase Array, Western blot, and/or immunohistochemistry.

Results: Samples derived from ocular rosacea patients showed increased levels of phosphorylated (active) p38 and Erk kinases. Phosphoproteins were mainly localized to the epidermis of affected eyelids.

Conclusions: This finding provides a novel potential therapeutic target for treatment of ocular rosacea and possibly other forms of rosacea. Further testing is required to determine if p38 and Erk activation have a causal role in ocular rosacea. The selective activation of keratinocytes in the affected skin suggests that topical pathway inhibition may be an effective treatment that will ultimately prevent ocular surface damage due to ocular rosacea.

Acne rosacea is a highly prevalent disease,1 and 58% to 72% of patients that suffer from this cutaneous ailment develop ophthalmic manifestations of their disease.2 These disorders are characterized by cutaneous irritation and cosmetic deformity, and the ocular variant of rosacea results in clinically significant surface disease, with subsequent dry eye syndrome, pain, blurred vision, tearing, and photophobia.39 
Several treatment options have been designed to address rosacea, and the multiplicity of therapies underscores the lack of efficacy of any particular one. Lifestyle modifications, eyelid hygiene, topical and oral medications, laser and light-based therapies, and surgical interventions have all been employed in the management of rosacea,10 although the results of these treatments have not been uniformly effective and this ailment remains incurable. In fact, our current modalities either address inflammation in a very general sense (i.e., corticosteroids, dietary modifications, nonsteroidal agents, antibiotics, and so on) or attempt to reverse existing damage distal to the site of the pathology (i.e., meibomian gland probing, corneal surgery to address perforations, and so on). Consequently, our current therapeutic armamentarium fails to tackle the immunologic and cellular aberrancies of this disease and thus cannot suppress rosacea in a highly targeted, specific fashion. 
Recently, several studies have advanced our comprehension of the biologic aberrancies inherent to rosacea.1012 In order to characterize the molecular biology of ocular rosacea, we previously assessed the concentrations of 48 individual cytokines, chemokines, and angiogenic factors in cutaneous biopsies of the disease and in control patients, and identified statistically significant enrichments of interleukins-1β and -16, stem cell factor, monocyte chemoattractant protein-1, and the monokine induced by interferon gamma.13 Given that these molecules have been previously associated with the innate immune system, follow-up studies were performed to better understand this process. We previously demonstrated an enrichment of Toll-like receptors in cutaneous biopsies of ocular rosacea14; these proteins provide constant surveillance against invading pathogens, and, upon stimulation, coordinate an innate immune response.15 Perhaps most excitingly, the number of Toll-like receptors correlated with the presence of the vascular abnormalities CD105 and intercellular-adhesion molecule-1 in cutaneous preparations of ocular rosacea, further implicating this variant of immunity in the disease.14 
The current study sought to further refine our understanding of the cellular biology of rosacea. In essence, previous studies have identified the role of Toll-like receptors in governing this disorder and have implicated key effector molecules that lead to its clinical manifestations. Careful analysis of the alteration of cell-signaling pathways that facilitate the development of rosacea may lead to the identification of discrete targets for highly specific therapeutic intervention in the management of rosacea and will ideally enhance our comprehension of the mechanisms that potentiate it. 
Methods
Patient Demographics
For the immunohistochemistry (six women and six men in each group), the average age for rosacea patients was 57.1 years (standard deviation = 6.9 years) and the average for controls was 56.4 years (standard deviation = 7.3 years). For the Western blots, the rosacea average was 74 years (standard deviation = 20.4 years) and the control average was 74.3 years (standard deviation = 16.3 years). The rosacea group had eight women and six men, and the control group had seven women and three men. For the profiler, the rosacea average was 72.75 years (standard deviation = 11.7 years) and the control average was 75.4 years (standard deviation = 13.6 years). The average ages of patients and controls were not statistically significantly different. 
Biopsies and Protein Extraction
We obtained patient informed consent to analyze cutaneous biopsies resulting from elective lower lid ectropion surgery. This protocol was reviewed and approved by the Albany Medical College Institutional Review Board, and adhered to the tenets of the Declaration of Helsinki. Immediately after resection of skin from patients with rosacea and from age- and sex-matched controls, the specimens were frozen at −80°C for later retrieval. A total of 31 samples were obtained. All patients underwent eyelid tightening, and patients with extensive lower eyelid malposition were specifically excluded, with the intent of eliminating potentially confounding chronic irritation and cicatrization. 
Each sample was thawed, weighed, and processed for protein extraction. For that, 300 μL ice-cold lysis buffer containing 1% Triton X-100 (Sigma-Aldrich Corp., St. Louis, MO, USA) in phosphate-buffered saline (PBS) containing protease and phosphatase inhibitor cocktails (Roche, Indianapolis, IN, USA) and 50 mM pervanadate (Sigma-Aldrich Corp.) was added to the sample together with 250 mg zirconia/silica beads (BioSpec Products, Bartlesville, OK, USA). Then samples were homogenized through three cycles of 1 minute each on a Mini-Beadbeater-96 (BioSpec Products). Samples were centrifuged at >12,000g for 2 minutes at 4°C and the supernatant was cleared by a second centrifugation for 15 minutes at >12,000 for 2 minutes at 4°C. Lysates were aliquoted. Some aliquots received 1× volume of 2× Laemmli buffer and were boiled for 5 minutes. Then, samples were immediately stored at −80°C until use. Sample identity was masked during all experimentation and unmasked only after all samples were quantified. 
Protein Arrays and Western Blot
Detection of 46 phosphoproteins was performed on Proteome Profiler Human Phospho-Kinase Array Kit membranes (R&D Systems, Minneapolis, MN, USA) according to manufacturer's instructions using 100 μg total protein. Western blots were performed using 10 μg/lane of total protein and detected using the primary antibodies detailed in Supplementary Table S1. Horseradish peroxidase–conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Signal was detected with West Pico or West Femto reagents (Pierce, Waltham, MA, USA) and a FujiFilm LAS-3000 imager (Tokyo, Japan). Band quantification was performed using FujiFilm MultiGauge software from raw image files according to manufacturer's instructions. 
Immunohistochemistry
Paraffin-embedded sections were deparaffinized and rehydrated with sequential steps in xylene and ethanol solutions (100% to 95% to 70%). Endogenous peroxidase activity was blocked with 0.5% H2O2/MeOH for 10 minutes at room temperature (RT) and antigen retrieval was done for 30 minutes at 100°C in 10 mM citrate buffer, pH 6. Samples were blocked with 5% normal goat serum (Vector S-1000, Burlingame, CA, USA) for 1 hour at RT. Primary antibodies were incubated at a dilution 1:80 in PBS overnight at 4°C. Biotinilated anti-mouse secondary antibodies (Vector ba-9200) 1:500 in PBS were incubated for 1 hour at RT. Then, samples were incubated with avidin/biotin peroxidase (Vector ABC kit Elite PK-6100) in the dark for 30 minutes at RT and signal was detected with 3,3′-diaminobenzidine (Immpact DAB; Vector SK-4105) and counterstained with hematoxylin (Vector H-3404) prior to dehydration and mounting with VectaMount (Vector H-5000). Positively stained cells were quantified per ×1000 field. Sample identity was masked during all experimentation and unmasked only after all samples were quantified. 
Statistical Analysis
Ocular rosacea versus control samples were compared by Student's t-test. A P < 0.05 was considered statistically significant. 
Results
Rosacea is a cutaneous ailment. The involvement of meibomian glands and ocular surface observed in ocular rosacea may be secondary to the effects of the cutaneous inflammation. In order to identify the mechanisms underlying ocular rosacea, we sought to compare the level of activation of multiple signaling pathways in diseased and control skin tissue by analyzing cutaneous biopsies resulting from elective lower lid ectropion surgery from patients with or without rosacea. The diagnosis of rosacea was made clinically through slit-lamp investigation. All patients in the rosacea group had evidence of bilateral chronic blepharitis, eyelid margin telangiectases, and meibomian gland clogging, in addition to the characteristic external cutaneous findings of the disease. All patients showed mild to moderate ocular rosacea; no severe cases of ocular rosacea were analyzed for this study. 
The initial pathway characterization was performed in eight samples using an unbiased approach consisting in the simultaneous analysis of 46 proteins. Lysates were allowed to bind to Proteome Profiler Human Phospho-Kinase Array Kit membranes (R&D Systems). Results are summarized in the Table. Among other signals, the levels of activation of p38 and Erk1/2 were increased in samples from patients with ocular rosacea when compared to samples from control patients without ocular rosacea (Fig. 1). 
Table
 
Human Phospho-Kinase Array Comparison Between Control and Rosacea Samples
Table
 
Human Phospho-Kinase Array Comparison Between Control and Rosacea Samples
Figure 1
 
Phosphoprotein profiler array data showing increased phosphorylation of p38 and Erk kinases in eyelid biopsies from patients with ocular rosacea compared to age-matched controls. *P < 0.05; #P < 0.1 (2-tailed Student's t-test, n = 4 controls and 4 rosacea eyelids).
Figure 1
 
Phosphoprotein profiler array data showing increased phosphorylation of p38 and Erk kinases in eyelid biopsies from patients with ocular rosacea compared to age-matched controls. *P < 0.05; #P < 0.1 (2-tailed Student's t-test, n = 4 controls and 4 rosacea eyelids).
To confirm the results obtained by the phosphokinase array, we tested the levels of pT180Y182-p38 (p-p38) and pT202Y204-Erk1 (p-Erk) in the proteins extracted from these and other samples by Western blot analysis. Figure 2A shows a sample of the band pattern obtained by the Western blots, while Figure 2B shows the quantification of the data. Western blot and band quantification was performed by a researcher masked to sample diagnoses. Confirming the results obtained previously, we observed a significant increase in the levels of p-p38 and p-Erk1/2 in rosacea samples. Other candidate signals were significantly increased in the proteome profiler array (CREB, Stat5a, Fyn, Yes, Rsk1/2/3, and HSP60), but subsequent analyses by Western blot were unable to confirm those results (Supplementary Fig. S1). 
Figure 2
 
Western blot analysis to confirm the initial results obtained with the protein profiler arrays. (A) Sample Western blot bands corresponding to the phosphorylated and total forms of each protein. (B) Band quantification expressed as a normalized result (p-protein/total protein). *P < 0.05 (1-tailed Student's t-test, n = 10 controls and 14 rosacea eyelids).
Figure 2
 
Western blot analysis to confirm the initial results obtained with the protein profiler arrays. (A) Sample Western blot bands corresponding to the phosphorylated and total forms of each protein. (B) Band quantification expressed as a normalized result (p-protein/total protein). *P < 0.05 (1-tailed Student's t-test, n = 10 controls and 14 rosacea eyelids).
We then sought to confirm these results with a different technique and to determine which cell type(s) within the skin displayed increased p-p38 and p-Erk1/2 levels. Immunohistochemical analysis was performed on paraffin-embedded sections obtained from archived eyelid biopsies. As with previous assays, we compared lower eyelid ectropion biopsies from patients with or without ocular rosacea. Immunohistochemical staining and positive cell quantification were performed by two researchers masked to sample diagnoses. As shown in Figure 3A, the signal for these two activated protein species was almost exclusively located within the epidermis, suggesting that the keratinocyte is the main cell type responsible for the observed differences in the tissue lysates. Consistent with the overall increase of phosphorylated p38 and Erk observed in the protein array and Western blot assays, the numbers of epithelial cells that stained positively for these cell signals were statistically significantly higher in cutaneous biopsies of ocular rosacea than in age- and sex-matched controls (Fig. 3B). 
Figure 3
 
Immunohistochemical localization of p-p38 and p-Erk. (A) Representative fields showing the marked epidermal localization of the phosphorylated proteins in the rosacea samples. (B) Quantification of the number of positive cells/field. *P < 0.05 (2-tailed Student's t-test, n = 12 controls and 12 rosacea eyelids).
Figure 3
 
Immunohistochemical localization of p-p38 and p-Erk. (A) Representative fields showing the marked epidermal localization of the phosphorylated proteins in the rosacea samples. (B) Quantification of the number of positive cells/field. *P < 0.05 (2-tailed Student's t-test, n = 12 controls and 12 rosacea eyelids).
Discussion
Despite the epidemiologically rich nature of rosacea and its significant impact on the lives of patients that suffer from it, our current treatment options are woefully inadequate.10 In fact, our treatment options are generally nonspecific and do not target the cellular and immunologic features that distinguish the disease from normal, healthy skin. As such, enhancements in our comprehension of the biology of rosacea will facilitate the development of new highly specific interventions in a highly translational manner. Ocular rosacea is a skin disease, and ocular surface and meibomian gland involvement are thought to be secondary to the inflammatory signaling emanating from the eyelid and periocular skin. Thus, we focused on our studies to assess the changes in eyelid skin intracellular signaling pathways to evaluate a potential cause, rather than secondary outcomes, of ocular rosacea. 
The current study is the first to implicate mitogen-activated protein kinase (MAPK) activation in the pathogenesis of this disorder. Specifically, based on analyses that employed multiple different mechanisms to assess the enrichment of a wide array of individual kinases, activation of p38 and Erk was clearly increased in the epidermis of eyelids with ocular rosacea. Due to the study design, only mild to moderate cases were analyzed, and thus whether the intensity of activation correlates with disease severity is still an open question. A potential limitation of findings based on highly multiplexed assays is the increased chance of false-positive and -negative results. Our findings that both p38 and Erk pathways are activated in rosacea are, however, highly unlikely to derive from nonspecific interactions, as they were consistently observed using three different methodologies: chemiluminescent membrane-based antibody array and Western blot of Triton X-100 lysates, as well as immunohistochemistry of paraffin-embedded tissue sections. 
Due to the lack of available animal models of rosacea, it is very difficult to test a causal relationship for any intracellular signal in preclinical studies. However, our findings are consistent with the existing literature regarding cutaneous inflammation, and a few studies have explored the role that these proteins play in dermatologic disease. Yamasaki et al.16 showed that an increase in the cathelicidin LL37, an antimicrobial peptide, promoted skin inflammation in rosacea. More recently, the same group reported that p38 inhibition selectively prevented the induction of IL-36γ in keratinocytes that were challenged in vitro with LL37.17 Moreover, treatment with the anti-inflammatory agent azelaic acid (a known topical therapy for acne that has been also successfully used to treat some patients with rosacea18) reduced the expression of cathelicidin in mouse skin and serine protease activity in the skin of patients with rosacea.19 It is well established that ultraviolet light (UVL), a trigger of rosacea flares, induces p38 activation in keratinocytes.20 Importantly, Mastrofrancesco and colleagues21 demonstrated that treatment with azelaic acid inhibited the UVL-induced increase in p38 phosphorylation in keratinocytes in vitro. Thus, the published literature in vitro suggests a role for p38 in the mechanisms of cathelicidin-mediated skin inflammation. Nonetheless, we are unaware of any previous studies that have directly assessed the levels of p38 and other MAPKs in cutaneous biopsies of rosacea; and, to our knowledge, this study is the first to directly implicate these proteins in the pathogenesis of this disorder. 
Along with other studies, our previous investigations have implicated Toll-like receptors (TLR) in the pathogenesis of rosacea.14,22 These receptors are well known to promote p38 and Erk activation,23,24 and these pathways have been shown to mediate multiple keratinocyte responses. For example, TLR2-mediated p38 and Erk activation was required for the inflammatory and antimicrobial responses of keratinocytes challenged with streptococcal M1 protein,25 Staphylococcus epidermidis LP01 lipopeptide,26 or Candida albicans phospholipomannan.27 Moreover, the TLR4/p38 signaling axis is essential for normal cutaneous wound healing,28 and expression of TLR2 and TLR4 was found to be altered in atopic dermatitis, contact dermatitis, and psoriasis.29 The identification of the increased activation of p38 and Erk signaling in diseased skin suggests an active TLR/p38 axis as an important mechanism of ocular rosacea. This activation may ultimately yield increased cytokine levels, which then may result in the clinical manifestations of rosacea. 
The activation of a cutaneous Erk pathway may also have a role in rosacea pathogenesis. For example, rosacea can promote a thickening of the epidermis. This is particularly evident in the phymatous subtype in which multiple layers of skin may lead to gross changes in cheeks and nose.30 The Erk pathway has long been associated with epidermal proliferation and is an essential mediator of multiple growth factors, including epidermal growth factor.3133 Another typical hallmark of rosacea skin is the presence of multiple telangiectases.30 Erk activation was shown to be critical in an in vitro model of activin receptor-like kinase 1 hereditary hemorrhagic telangiectasia (HHT).34 Further, Erk is a key mediator of vascular endothelial growth factor,35 another key factor in the pathogenesis of HHT.3638 It is unknown whether Erk activity is involved in the development of cutaneous telangiectasia observed in rosacea skin. The roles of Erk signaling in the epidermis appear to be complex, as this pathway is required to prevent allergic skin disease in mice,39 and a common side effect of the pathway inhibitor trametinib is skin rash.40,41 
Selective inhibition of p38 and Erk pathways represents an intriguing possibility for the management of ocular rosacea. The absence of targeted remedies for this disease strongly indicates that cellular therapies would be a welcome addition to our interventional armamentarium, and mechanisms that target the disease at the site of its pathology (i.e., inflamed skin) may prevent and heal ocular surface damage. In fact, multiple clinical trials are currently active to test safety and efficacy of p38 inhibitors in Langerhans cell histiocytosis42 as well as multiple inflammatory diseases,4345 including chronic obstructive pulmonary disease,46 cardiovascular disease,43 and rheumatoid arthritis.47 Targeting MAPK signaling has been shown to have an acceptable safety profile. A recently completed large phase III trial to study the ability of Losmapimod (a selective p38 inhibitor) to reduce the incidence of cardiovascular events in subjects with acute coronary syndrome (LATITUDE-TIMI 60, NCT02145468) showed a good safety profile.48 Similarly, MEK1/2 (the upstream activator of Erk1/2) inhibitors are being currently tested in phase III trials for the treatment of multiple oncologic pathologies,49,50 and Trametinib, a MEK1/2 inhibitor, has been approved for the treatment of melanoma containing BRAFV600E or V600K mutations.40,50 While other fields have adopted kinase inhibitors, the use of cellular therapies in the treatment of rosacea is a novel, highly exciting opportunity to enhance the lives of patients that suffer from this currently incurable disease. By documenting a constitutive increase of p38 and Erk activation in this disease, we therefore propose the possibility of the use of agents that inhibit these cell signals in the treatment of rosacea. Further studies are required to determine a possible involvement of these pathways in other aspects of this disease, such as meibomian gland dysfunction. 
Specific activation of p38 and Erk pathways in the epidermis of eyelid skin with ocular rosacea suggests the possible involvement of these kinases in the pathogenesis of the disease. While further studies are required to determine a causal role for these kinases, the epidermal activation suggests that topical inhibition of these kinases might be a potential avenue to specifically treat ocular rosacea. 
Acknowledgments
Supported by a grant from the Sight Society of Northeastern New York (to APA) and by an Albany Medical Center Institutional Seed Grant (to EJW and APA). The sponsors did not have any role in the design, execution, or publication of this research. 
Disclosure: E.J. Wladis, Praxis Biotechnology, Inc. (I), P; S. Swamy, None; A. Herrmann, None; J. Yang, None; J.A. Carlson, None; A.P. Adam, Praxis Biotechnology, Inc. (I), P 
References
Tan J, Berg M. Rosacea: current state of epidemiology. J Am Acad Dermatol. 2013; 69: S27–S35.
Vieira AC, Hofling-Lima AL, Mannis MJ. Ocular rosacea - a review. Arq Bras Oftalmol. 2012; 75: 363–369.
Bron AJ, Tiffany JM. The contribution of meibomian disease to dry eye. Ocul Surf. 2004; 2: 149–165.
Ramamurthi S, Rahman MQ, Dutton GN, Ramaesh K. Pathogenesis, clinical features and management of recurrent corneal erosions. Eye (Lond). 2006; 20: 635–644.
Afonso AA, Sobrin L, Monroy DC, Selzer M, Lokeshwar B, Pflugfelder SC. Tear fluid gelatinase B activity correlates with IL-1alpha concentration and fluorescein clearance in ocular rosacea. Invest Ophthalmol Vis Sci. 1999; 40: 2506–2512.
Jenkins MS, Brown SI, Lempert SL, Weinberg RJ. Ocular rosacea. Am J Ophthalmol 1979; 88: 618–622.
Akpek EK, Merchant A, Pinar V, Foster CS. Ocular rosacea: patient characteristics and follow-up. Ophthalmology. 1997; 104: 1863–1867.
Pflugfelder SC. Hormonal deficiencies and dry eye. Arch Ophthalmol. 2004; 122: 273–274.
Oltz M, Check J. Rosacea and its ocular manifestations. Optometry. 2011; 82: 92–103.
Wladis EJ, Adam AP. Current and emerging therapies for ocular rosacea. US Ophthalmic Rev. 2013; 6: 86–88.
Del Rosso JQ, Thiboutot D, Gallo R, et al. Consensus recommendations from the American Acne & Rosacea Society on the management of rosacea, part 1: a status report on the disease state, general measures, and adjunctive skin care. Cutis. 2013; 92: 234–240.
Steinhoff M, Schauber J, Leyden JJ. New insights into rosacea pathophysiology: a review of recent findings. J Am Acad Dermatol. 2013; 69: S15–S26.
Wladis EJ, Iglesias BV, Adam AP, Gosselin EJ. Molecular biologic assessment of cutaneous specimens of ocular rosacea. Ophthal Plast Reconstr Surg. 2012; 28: 246–250.
Wladis EJ, Carlson JA, Wang MS, Bhoiwala DP, Adam AP. Toll-like receptors and vascular markers in ocular rosacea. Ophthal Plast Reconstr Surg. 2013; 29: 290–293.
Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harb Perspect Biol. 2012; 4: a006049.
Yamasaki K, Di Nardo A, Bardan A, et al. Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea. Nat Med. 2007; 13: 975–980.
Li N, Yamasaki K, Saito R, et al. Alarmin function of cathelicidin antimicrobial peptide LL37 through IL-36gamma induction in human epidermal keratinocytes. J Immunol. 2014; 193: 5140–5148.
Jones DA. Rosacea, reactive oxygen species, and azelaic acid. J Clin Aesthet Dermatol. 2009; 2: 26–30.
Coda AB, Hata T, Miller J, et al. Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel. J Am Acad Dermatol. 2013; 69: 570–577.
Muthusamy V, Piva TJ. The UV response of the skin: a review of the MAPK, NFkappaB and TNFalpha signal transduction pathways. Arch Dermatol Res. 2010; 302: 5–17.
Mastrofrancesco A, Ottaviani M, Aspite N, et al. Azelaic acid modulates the inflammatory response in normal human keratinocytes through PPARgamma activation. Exp Dermatol. 2010; 19: 813–820.
Yamasaki K, Kanada K, Macleod DT, et al. TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes. J Invest Dermatol. 2011; 131: 688–697.
Miller LS. Toll-like receptors in skin. Adv Dermatol. 2008; 24: 71–87.
Oviedo-Boyso J, Bravo-Patino A, Baizabal-Aguirre VM. Collaborative action of Toll-like and NOD-like receptors as modulators of the inflammatory response to pathogenic bacteria. Mediators Inflamm. 2014; 2014: 432785.
Persson ST, Wilk L, Morgelin M, Herwald H. Vigilant keratinocytes trigger pathogen-associated molecular pattern signaling in response to streptococcal M1 protein. Infect Immun. 2015; 83: 4673–4681.
Li D, Lei H, Li Z, Li H, Wang Y, Lai Y. A novel lipopeptide from skin commensal activates TLR2/CD36-p38 MAPK signaling to increase antibacterial defense against bacterial infection. PLoS One. 2013; 8: e58288.
Li M, Chen Q, Tang R, Shen Y, Liu WD. The expression of beta-defensin-2, 3 and LL-37 induced by Candida albicans phospholipomannan in human keratinocytes. J Dermatol Sci. 2011; 61: 72–75.
Chen L, Guo S, Ranzer MJ, DiPietro LA. Toll-like receptor 4 has an essential role in early skin wound healing. J Invest Dermatol. 2013; 133: 258–267.
Panzer R, Blobel C, Folster-Holst R, Proksch E. TLR2 and TLR4 expression in atopic dermatitis, contact dermatitis and psoriasis. Exp Dermatol. 2014; 23: 364–366.
Wilkin J, Dahl M, Detmar M, et al. Standard grading system for rosacea: report of the National Rosacea Society Expert Committee on the classification and staging of rosacea. J Am Acad Dermatol. 2004; 50: 907–912.
Ratushny V, Gober MD, Hick R, Ridky TW, Seykora JT. From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma. J Clin Invest. 2012; 122: 464–472.
Kern F, Niault T, Baccarini M. Ras and Raf pathways in epidermis development and carcinogenesis. Br J Cancer. 2011; 104: 229–234.
Pastore S, Mascia F, Mariani V, Girolomoni G. The epidermal growth factor receptor system in skin repair and inflammation. J Invest Dermatol. 2008; 128: 1365–1374.
David L, Mallet C, Vailhe B, Lamouille S, Feige JJ, Bailly S. Activin receptor-like kinase 1 inhibits human microvascular endothelial cell migration: potential roles for JNK and ERK. J Cell Physiol. 2007; 213: 484–489.
Aramoto H, Breslin JW, Pappas PJ, Hobson RWII, Duran WN. Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation. Am J Physiol. 2004; 287: H1590–H1598.
Giordano P, Lenato GM, Pierucci P, et al. Effects of VEGF on phenotypic severity in children with hereditary hemorrhagic telangiectasia. J Pediatr Hematol Oncol. 2009; 31: 577–582.
Epperla N, Hocking W. Blessing for the bleeder: bevacizumab in hereditary hemorrhagic telangiectasia. Clin Med Res. 2015; 13: 32–35.
Bose P, Holter JL, Selby GB. Bevacizumab in hereditary hemorrhagic telangiectasia. New Engl J Med. 2009; 360: 2143–2144.
Raguz J, Jeric I, Niault T, et al. Epidermal RAF prevents allergic skin disease. Elife. 2016; 5: e14012.
Flaherty KT, Robert C, Hersey P, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. New Engl J Med. 2012; 367: 107–114.
Welsh SJ, Corrie PG. Management of BRAF and MEK inhibitor toxicities in patients with metastatic melanoma. Ther Adv Med Oncol. 2015; 7: 122–136.
Arico M. Langerhans cell histiocytosis in children: from the bench to bedside for an updated therapy. Br J Haematol. 2016; 173: 663–673.
Fisk M, Gajendragadkar PR, Maki-Petaja KM, Wilkinson IB, Cheriyan J. Therapeutic potential of p38 MAP kinase inhibition in the management of cardiovascular disease. Am J Cardiovasc Drugs. 2014; 14: 155–165.
Arthur JS, Ley SC. Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol. 2013; 13: 679–692.
Martin ED, Bassi R, Marber MS. p38 MAPK in cardioprotection - are we there yet? Br J Pharmacol. 2015; 172: 2101–2113.
Norman P. Investigational p38 inhibitors for the treatment of chronic obstructive pulmonary disease. Exp Opin Investig Drugs. 2015; 24: 383–392.
Salgado E, Maneiro JR, Carmona L, Gomez-Reino JJ. Safety profile of protein kinase inhibitors in rheumatoid arthritis: systematic review and meta-analysis. Ann Rheum Dis. 2014; 73: 871–882.
O'Donoghue ML, Glaser R, Cavender MA, et al. Effect of losmapimod on cardiovascular outcomes in patients hospitalized with acute myocardial infarction: a randomized clinical trial. JAMA. 2016; 315: 1591–1599.
Sullivan R, LoRusso P, Boerner S, Dummer R. Achievements and challenges of molecular targeted therapy in melanoma. Am Soc Clin Oncol Educ Book. 2015; 177–186.
Akinleye A, Furqan M, Mukhi N, Ravella P, Liu D. MEK and the inhibitors: from bench to bedside. J Hematol Oncol. 2013; 6: 27.
Figure 1
 
Phosphoprotein profiler array data showing increased phosphorylation of p38 and Erk kinases in eyelid biopsies from patients with ocular rosacea compared to age-matched controls. *P < 0.05; #P < 0.1 (2-tailed Student's t-test, n = 4 controls and 4 rosacea eyelids).
Figure 1
 
Phosphoprotein profiler array data showing increased phosphorylation of p38 and Erk kinases in eyelid biopsies from patients with ocular rosacea compared to age-matched controls. *P < 0.05; #P < 0.1 (2-tailed Student's t-test, n = 4 controls and 4 rosacea eyelids).
Figure 2
 
Western blot analysis to confirm the initial results obtained with the protein profiler arrays. (A) Sample Western blot bands corresponding to the phosphorylated and total forms of each protein. (B) Band quantification expressed as a normalized result (p-protein/total protein). *P < 0.05 (1-tailed Student's t-test, n = 10 controls and 14 rosacea eyelids).
Figure 2
 
Western blot analysis to confirm the initial results obtained with the protein profiler arrays. (A) Sample Western blot bands corresponding to the phosphorylated and total forms of each protein. (B) Band quantification expressed as a normalized result (p-protein/total protein). *P < 0.05 (1-tailed Student's t-test, n = 10 controls and 14 rosacea eyelids).
Figure 3
 
Immunohistochemical localization of p-p38 and p-Erk. (A) Representative fields showing the marked epidermal localization of the phosphorylated proteins in the rosacea samples. (B) Quantification of the number of positive cells/field. *P < 0.05 (2-tailed Student's t-test, n = 12 controls and 12 rosacea eyelids).
Figure 3
 
Immunohistochemical localization of p-p38 and p-Erk. (A) Representative fields showing the marked epidermal localization of the phosphorylated proteins in the rosacea samples. (B) Quantification of the number of positive cells/field. *P < 0.05 (2-tailed Student's t-test, n = 12 controls and 12 rosacea eyelids).
Table
 
Human Phospho-Kinase Array Comparison Between Control and Rosacea Samples
Table
 
Human Phospho-Kinase Array Comparison Between Control and Rosacea Samples
Supplement 1
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×