ORIGINAL ARTICLE Stat3 as a Therapeutic Target for the Treatment of Psoriasis: A Clinical Feasibility Study with STA-21, a Stat3 Inhibitor Ken Miyoshil Mikiro Takaishil Kimiko Nakajimal , Mitsunori Ikedal, Takashl Kandal, Masahito Tarutanil, Tatsuo liyama2, Naoki Asa03, John DiGiovanni4 and Shigetoshi Sanol Epidermal keratinocytes in psoriatic lesions are characterized by activated Stat3, and increased levels of cytokines and growth factors that promote Stat3 activation have been found within psoriatic lesions. K5.
Stat3C transgenic mice, in which keratinocytes express a constitutively active Stat3, develop soriasis-like skin lesions. In this study, we examined whether STA-21, a small Stat3 inhibitor could be useful in ameliorating S»ipeto n ut*ge the skin lesions not o or2g psoriasis. Treatment dependent nuclear tr slocat keratinocytes in vitro STA-21 in a dose-dep but also in human hibited the cytokine- ormal human on was inhibited by downregulation of c-Myc and cyclin DI, whereas involucrin, transglutaminase 1, and keratin 10 levels were upregulated. Topical application of STA-21 abolished the generation of skin lesions in K5.
Stat3C mice. Finally, we treated psoriasis patients with STA21 -containing ointment in a onrandomized study. Psoriatic lesions In Six of the eight patients showed improvement after topical STA-21 treatment for 2 weeks. Therefore, we conclude that targeting Stat3 may to nex: page may lead to a therapy for psoriasis. Journal of Investigative Dermatology (2011) 131, 108-117; doi: 10. 1038/jid. 2010. 255; published online 2 September 2010 INTRODUCTION Our previous study elucidated the role of Stat3 signaling in keratinocytes during the development of psoriatic lesions (sano et ala, 200%, 2008).
K5. Stat3C transgenic mice, in which Stat3 is constitutively active in keratinocytes, develop soriasiform lesions after wounding stimuli or topical treatment with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), which strongly suggests that Stat3 activation is required for the development of psoriasis. Previous studies have shown that the IL-23/lL-17 pathway is linked to the induction of a number of inflammatory diseases, including multiple sclerosis, arthritis, inflammatory bowel diseases, and psoriasis (Cua et al. , 2003; Murphy et al. Department of Dermatology’, Kochi Medical School, Kochi University, Okocho, Nankoku, Japan; 2Clinical Trial Center, Kochi Medical School, Kochi University, Okocho, Nankoku, Japan; 3Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan and 4 Department of Nutritional Sciences, Dell Pediatric Research Institute, The University of Texas at Austin, Austin, Texas, USA These authors contributed equally to this work Correspondence: Shigetoshi Sano, Department of Dermatology’, Kochi Medical School, Kochi University, Kohasu, Okocho, Nankoku 783-8505, Japan.
E-mail: sano. derma@kochi-u. ac. jp Abbreviations: HB-EGF, heparin-binding EGF-like growth factor; NHK, normal human keratinocyt Abbreviations: HB-EGF, heparin-binding EGF-like growth factor; NHK. normal human keratinocyte; TPA, 12-0- tetradecanoylphorbol-13-acetate Received 15 January 2010; revised 22 June 2010; accepted 1 uly 2010; published online 2 September 2010 2003; yen et al. , 2006; Wilson et al. , 2007). The most distinct evidence for the role of IL-23/Th1 7 in psoriasis comes from clinical studies.
A clinical study showed that anti-lL-12/ IL-23p40 therapy is an effective treatment for psoriasis (Krueger et al. 2007). Amelioration of psoriasis is associated with reduced Th17 responses (Zaba et al. , 2007; Haider et al. , 2008). 11_-22, cytokine produced from Th17, is elevated in the blood of psoriasis patients (Wolk et al. , 2006), and triggering the IL-22R mediates the proliferation and migration of keratinocytes via Stat3 activation (Boniface et al. , 2005).
Furthermore, Studies using gene-targeted mice and reconstituted human epidermis revealed that IL-22 mediates acanthosis through the activation of Stat3 in keratinocytes, which strongly suggests that IL-22, as an efector cytokine by Th17, mediares the cross-talk between immunocytes and keratinocytes in the pathogenesis of psoriasis (sano et al. , 2005a; sa et al. , 2007, Zheng et al. , 2007). Ir, addit,on to 11_-22, other 11-20 subfamily cytokines, such as 11_-19, 11_-20, 11_-24, and 11_-26, have been implicated in psoriasis and are able to phosphorylate Stat3 in keratinocytes (Blumberg et al. 2001; Romer et al. , 2003; Boniface et ala, 2005; Sa et al. , 2007; Tohyama et al. , 2009; wolk et al. , 2009). Furthermore, EGF family growth factors, including hep 2007; Tohyama et al. , 2009; Wolk et al. , 2009). Furthermore, EGF family growth factors, including heparin-binding EGFIike growth factor (HB-EGF), TGF-a, and amphiregulin, have also been suggested to be important (Piepkorn, 19%; Piepkorn et al. 1998). These EGF family growth factors are produced by keratinocytes as autocrine/juxtacrine factors that induce Stat3 activation (Sartor et al. 1997; sano et al. , 1999; T0kumaru et al. , 2005). Taken together, Stat3 has a major & 2011 The Society for Investigative Dermatology 108 Journal of Investigative Dermatology (201 1), Volume 131 K Miyoshi et al. Stat3 as a Therapeutic Target Psoriasis Uninvolved Lesion Normal skin Stat3/DAPl Relative mRNA expression of psoriatic lesion to uninvolved skin (normalized with HPRT) 10,000 1,000 100 10 from patients with psoriasis (n 1/4 5). IL-20 subfamily cytokines and HB-EGF mRNAs were increased in the lesional skin, but 11–4 mRNA was not increased. unction in the signal transduction of psoriatic keratinocytes in response to cytokine/growth factors. This notion supports the idea that the use of Stat3 inhibitors could be an effective therapy for psoriasis. We showed previously that topical treatment of K5. Stat3C transgenic mice with Stat3 decoy oligonucleotides inhibits the de novo generation of psoriatic-like lesions and attenuates the epidermal hyperplasia of preexisting lesions (Sano et al. , 2005a). However, as decoy oligonucleotides are too arge to be absorbed epicutaneously, we have searched for Small Stat3 inhibitors suitable for topical application.
Song et al. (2005) identified an antibiotic termed STA-21 (or ochromycinone) as a Stat3 inhibitor, which blocks Stat3 dimerization and DNA binding, and in this study we tested the efficacy of STA-21 in treating psoriatic lesions. RESULTS Stat3 activation in epidermal keratinocytes and increased expression of Stat3-stimulatory ligands in psoriatic lesions Immunohistochemistry with an anti-Stat3 antibody revealed that epidermal keratinocytes in psoriatic lesions show an intense Stat3 ignal in nuclei (Figure la), indicating Stat3 activation as previously reported (Sano et al. , 2005a).
However, Stat3 expression was obscure in the epidermis of uninvolved skin from psoriasis patients and from normal controls. It has been shown that cytokines (e. g. , 11_-20 subfamily cytokines) and growth factors (e. g. , EGF subfamily gro s OF that cytokines (e. g. , I -20 subfamily cytokines) and growth factors (e. g. , EGF subfamily growth factors) that cause Stat3 activation are upregulated in psoriasis (Cook et ala, 1997; Blumberg et al. , 2001 Boniface et al. , 2007; Nickoloff, 2007; sa et al. 2007, Zheng et al. 2007). We verified the transcriptional levels of mRNAs www. jidonline. rg 109 NO GF DMSO EGF IL-6 IL-22 STA-21 3. 0 Relative cell number 2. 5 2. 0 1. 5 1. 0 0. 5 0. 0 0 24 (h) 48 72 DMSO 2. 5 120 100 Cytotoxicity 80 60 40 20 0 ax M ag M SO en 5 10 2040 2. 5 20 6 OF Cytotoxicity assay of STA-21 in vitro. Lactate dehydrogenase (LDH) is measured using the supernatants of NHK cultured for 24 hours in the presence of STA-21 at the indicated concentrations. NHK in DMSO (02%) alone and freeze-thaw control are set at O and 100% of LDH release, respectively. Data are means±SD from triplicate cultures. d) Downregulation of c-Myc and cyclin DI mRNA in NHK by STA-21 (20 mM).
Relative transcript levels measured by real-time RT-PCR of the relative expression from NHK cultured in the presence of STA-21 for 24 hours (shaded bars) and for 48 hours (dotted bars) compared with untreated cells (black bars) are shown. A representative result from three independent experiments is shown. encoding these cytokines in psoriatic lesions. Real-time RTPCR revealed that Stat3-stimulating ligands, including 11_-22, 11_-20, IL-1 9, and HB-EGF, were upregulated in the involved skin compared with the uninvolved skin, whereas IL-4 was not ncreased (Figure lb).
These results suggest that these molecules cause persistent Stat3 activation of keratinocytes that could give rise to psoriatic changes. STA-21 inhibits the nuclear translocation of Stat3 and the proliferation of normal human keratinocytes in vitro STA-21 (ochromycinone) is a known antibiotic and was identified as a Stat3 inhibitor through virtual screening by 110 Journal of Investigative Dermatology (201 1), Volume 131 Song et al. (2005). STA-21 hinders Stat3 dimerization and its nuclear translocation in human carcinoma cells, which constitutively express activated Stat3 (Song et ala, 2005).
We examined wheth carcinoma cells, which constitutively express activated Stat3 (Song et al. , 2005). We examined whether STA-21 inhibits Stat3 signaling in normal human keratinocytes (NHK) as well. Stimulation of NHK WIth EGF, 11–5, or 11–22 caused a rapid nuclear translocatlon of Stat3 (Figure 2a, upper panels). However, the addition of STA-21 to NHK inhibited the liganddependent nuclear translocation of Stat3 (Figure 2a, Iower panels). This result indicates that STA-21 efficiently inhibits Stat3 signaling in NHK as well as in cancer cell lines (Song et al. 2005). The proliferation of NHK in culture stimulated STA-21 (PM) D with EGF was inhibited by STA-21 in a dose-dependent manner (Figure 25), whereas a significant increase in cytotoxicity of STA-21 at the given concentrations was not found (Figure 2c). Both cyclin DI and c-Myc are Stat3 transcriptional targets and promote cell cycle progression. STA-21 downregulated cyclin DI and c-Myc at the transcriptional (Figure 2d) and protein levels (Figure 3c), which was associated with an antiproliferative effect of STA-21.
STA-21 upregulates involucrin, transglutaminase 1, and keratin 10 of NHK in vitro No reagent (calcitoriol) (Figure 3a), known stimuli for in vitro keratinocyte ifferentiation (Smith et al. , 1986; Pillai et al. , 1990; Takahashi et al. , 2003). This notion prompted us to examine whether STA-21 also induced keratinocyte differentiation markers in vitro as well. STA-21 elevated the levels of mRNAs encoding involucrin, transglutaminase 1, and keratin 10 but not keratin 14 (Figure 3b).
Western blotting analysis showed that STA-21 increased protein levels of involucrin and transglutaminase 1, which is similar to the effects of calcium and 1 ,25 (OH) vitamin D (calcitoriol) (Figure 3c). A densitometric analysis of the western blots revealed that STA-21 increased levels of involucrin and transglutaminase 1 proteins up to 1. 5- and 7. 6-fold, respectively, compared to the DMSO-treated controls. In contrast, STA-21 decreased levels of c-Myc and cyclin DI proteins to 0. 5- and 0. 4-fold, respectively, compared to the controls.
These changes correlated well with the changes in mRNA levels (Figure 2d). Previous Studies have shown that IL-22, which is a Th17 cytokine and induces Stat3 activation of keratinocytes, is critical in the development of psoriasis through promotion of keratinocyte proliferation and downregulation of differentiation (Boniface et al. , 2005; Wolk et al. 2006; Sa et al. , 2007; Nograles et ala, 2008). Here, we also verified the inhibitory effect of IL-22 on the expression of KIO and involucrin in cultured NHK (Figure 3d).
However, simultaneous inclusion with STA-21 reversed the effect of IL_22 and even increased their expression over the basal levels, suggesting th effect of IL22 and even increased their expression over the basal levels, suggesting that 11–22-induced downregulation ofthe differentiation markers might be attributed to Stat3 signaling. In VIVO inhibition of Stat3 nuclear translocatlon of epidermal keratinocytes by topical treatment with STA-21
Relative mRNA expression (normalized with HPRT) 25 20 15 105 Involucrin TGase 1 Keratin 10 Keratin 14 Stat3 c-Myc CyclinD1 Bcl-xL Involucrin TGase1 P-Actin SO STA21 ntca2+ no I ge 121086420 entST-2A-2 21 aglL-22relL al Our previous study showed that abrogation of Stat3 function by a decoy oligonucleotide inhibits the onset and reverses the bona fide psoriasiform lesions in K5. Stat3C mice, in which keratinocytes express a constitutively active Stat3. It should be noted that, compared with wild-type mice, K5. Stat3C mice constitutively showed incr clear localization, which was further increased foll pping (Figure 4a), which