TNIP1 Plays An Important Role In Proliferation Of Keratinocytes And Severer Imiquimod (IMQ)-induced Psoriasis-like Dermatitis In Mouse Model~*

Background:Psoriasis Vulgaris (referred as psoriasis in the following) is one kind of chronic inflammatory skin disease, which affects 1-2% European and North American population and 0.47% of Chinese population, and is characterized by abnormal proliferation and differentiation of epidermal cells, infiltration of immune cells and the increase of skin angiogenesis in histological changes. Clinically it predominantly affects the skin, presenting scaling erythema and plaque, accompanied by or not by itching, while the treatment is limited. Therefore psoriasis may impair the quality of life and constitute a heavy economic burden on patients. While the etiology is quite unknown, environmental and genetic factors are both confirmed involving in the pathogenesis of psoriasis. Recent genome-wide association studies (GWAS) have identified multiple genes as susceptibility genes of psoriasis, including the TNIP1 gene, which encodes the TNF-a-induced protein 3-interacting protein 1 (TNIP1). And the TNIP1 messenger ribonucleic acid (mRNA) level has been revealed to be increased in psoriatic skin.TNIP1, as well as other two family members, TNIP2 and TNIP3, all belong to TNIPs family. This family has earned its name by its capability to bind with A20, an ubiquitin-editing nuclear factor-κB (NF-κB) inhibitor protein, and have a common role in inhibition of NF-κB transcriptional activity, though limited sequence homology was observed among the family members. TNIP1 is an ubiquitin-binding protein, and distributes widely in different tissues/organs and different cell types. It distributes only in cytoplasma of most cell types, while locates in both cytoplasma and nucleus of keratinocyte. Moreover, TNIP1 expression showed different tendency in cancers of different origins, suggesting TNIP1 plays different or even contrary roles in different tissue/cell types. Besides A20 and NF-κB, TNIP1 also could work by targeting extracellular signal-regulated kinase1/2 (Erk1/2), peroxisome proliferator-activated receptors (PPARs), retinoic acid receptor-α and -γ (RAR-α/-γ), and CCAAT/enhancer-binding protein β (C/EBPβ). These distinctly different targets suggest that TNIP1 may have broad physiological and pathological functions, including aniti-inflammation, anti-apoptosis and sustain embryo development. As well known, the abnormal proliferation and differentiation of keratinocyte is predominant in psoriatic skin. And the upregulated expression of proinflammatory cytokines and chemokines by hyperproliferative epidermal cells may affect the clinical manifestation of psoriasis. Moreover, keratinocyte dysfunction is sufficient to initiate psoriasis-like inflammation on mice skin by abrogating activation protein 1 (AP1) pathway in keratinocyte signaling. A20, as well as TNIP2, has been shown to be able to modulate cell growth. But till now little is known about whether or how TNIP 1/TNIP1 playing in the pathogenesis of psoriasis.Objective:This experiment made pro-proliferative effect of TNIP 1 as the breakthrough point to explore the possible role of TNIP 1 in the pathogenesis mechanism of psoriasis, as TNIP1 gene has been identified as one of the susceptibility genes of psoriasis. And a specific small interfering hairpin RNA (shRNA) against TNIP1 (TNIP1 shRNA) or a recombinant TNIP1 (rTNIP1) was employed to alter the protein level of TNIP1 in keratinocyte. Studies were performed to confirm whether altered TNIP1 had effect on keratinocyte proliferation. Further studies verified whether TNIP1 work partially through Erk1/2 or C/EBPβ to modulate the synthesis of proliferation marker cytokeratin 6 (CK6). Finally, it intended to mimic downregulation of TNIP1 in vivo by intradermal injection of TNIP1 shRNA into mice model, and induced the mice developing into more severe psoriatic-like dermatitis by IMQ treatment.The main experimental methods and results:Part I Human skin lesions of moderate-to-severe plaque psoriasis were collected to detect the mRNA level and protein level of CK6 and TNIP1 by Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (n=12) and western bloting (n=4), respectively. We found the CK6 mRNA level (2.7-fold increase) and protein level (3.8-fold increase) both increased in psoriatic skin when compared with normal skin; the TNIP1 mRNA level increased (5.7 fold increase) while the protein level decreased (2- fold reduction) in psoriatic skin. And the decreased TNIP1 protein level in psoriatic skin was further confirmed by immunohistochemistry (IHC), which also demonstrated that TNIP1 protein distributed in both cytoplasma and nucleus of epidermal cells. Immunofluorescence staining of TNIP1 and confocal microscopy were performed to confirm that human keratinocyte cell line HaCaT cell constitutionally expressed endogenous TNIP1, distributing both in cytoplasm and nucleus of HaCaT. Primary human keratinocyte (PHK) were isolated and cultivated in vitro. And immunofluorescence staining of pan-anticytokeratin (AE1/AE3) and confocal microscopy were conducted to identify the primary cells we obtained were keratinocytes. As the immunofluorescence staining showed, green fluorescence was observed in the cytoplasma of all the cultivated PHKS after incubated in 10% fetal bovine serum (FBS) for 12 hours. Green fluorescent protein (GFP)-tagged lentiviral vectors experessing TNIP1 shRNA, rTNIP1 or Con shRNA were constructed and then used to infect HaCaT or PHKs. The stably infected cells were named as TNIP1 shRNA HaCaT/PHK, rTNIP1 HaCaT/PHK or Con shRNA HaCaT/PHK. And qRT-PCR and western bloting were performed to detect the mRNA level and protein level of TNIP1, respectively, in these cells of different groups. And it has been confirmed that TNIP1 shRNA effectively downregulated the mRNA level (2.5-fold decrease) and protein level (4.8-fold decrease) of TNIP 1 in keratinocyte, and rTNIP1 successfully upregulated TNIP1 mRNA level (2.3-fold increase) and protein level (9.6-fold increase) in keratinocyte. For in vivo studies,8-10 weeks, male, weighing about 25g of BALB/c mice were randomly divided into three groups:TNIP1 shRNA mice treated with intradermal injection of red fluorescent protein (RFP)-tagged TNIP1 shRNA lentiviral particles (TNIP1 shRNA mice), Con shRNA mice treated by RFP-tagged Con shRNA intradermal injection (Con shRNA mice) and normal mice without any treatment. A whole-body optical imaging system was used to detect the red fluorescence on mice skin seven days after intradermal injection. Then the injected area of mice skin was removed for IHC staining for IκBα, interleukin (IL)-1a, IL-1b and tumor necrosis factor a (TNFa), and for western bloting to detect TNIP1 protein level. The TNIP1 protein level in injection area of TNIP1 shRNA mice was downregulated to 55% of Con shRNA mice by intradermal injection of 7.5×107 TU lentiviral particles as the result of western bloting showed. And IHC staining showed that knockdown of TNIP1 in mice skin led to downregulation of IκBα, upregulation of IL-lb, but no obvious effect on IL-la and TNFa. Thus we have constructed the proper TNIP1-altered cell model and mice model in Part I.Part II Three different kinds of cell proliferation assays were performed to evaluate the proliferative level of TNIP1-altered HaCaT compared with Con shRNA HaCaT. One was to detect the mRNA level and protein level of CK6, the proliferation marker of keratinocyte in different groups; one was Cell Counting Kit-8 (CCK-8) assay, which reflected the viable counts of cells; and the other is enzyme-linked immuno sorbent assay (ELISA) BrdU assay, which directly indicated the cell proliferation level by determining the quantity of BrdU incorporated into cells. The CK6 mRNA level was 114% of control and protein level was 144% of control in TNIP1 shRNA HaCaT cells. And in rTNIP1 HaCaT cells, the CK6 mRNA level was 52% of control and protein level was 56% of control. The results of cell counting kit-8 (CCK8) assay revealed the proliferation rate of rTNIP1 HaCaT and TNIP1 shRNA HaCaT is 78% and 120% of Con shRNA HaCaT, respectively. The results of BrdU showed that the proliferation of TNIP1 shRNA HaCaT was 136% of control while the proliferation of rTNIP1 HaCaT was only 67% of control. All the results supported that the lower TNIP1 expression promoted the cell proliferation while the overexpression of TNIP1 suppressed the proliferation of keratinocyte. Similiar methods, CCK8 assay and BrdU incorporation assay, were used to confirm these results in PHKs. And the CK6 protein level in PHKs, detected by western bloting, was found to be concomitant with the proliferation rate.Part Ⅲ Western bloting was performed to detect the protein level of C/EBPβ, total Erkl/2 (t-Erkl/2) and phosphorylation of Erkl/2 (p-Erkl/2), and revealed that the level of C/EBPβ and p-Erkl/2 increased to 214% and 248% of control, respectively, in TNIP1 shRNA HaCaT/PHK cells, while decreased to 55% and 50% of control, respectively, in rTNIP1 HaCaT/PHK cells when compared with Con shRNA HaCaT/PHK. Co-Immunoprecipitation (Co-IP) was performed in HaCaT cells to verify that TNIP1 interacted with Erk2. Therefore C/EBPβ and Erkl/2 were selected as the possible intermediators to explore how altered TNIP1 affect the expression level of CK6. Three different specific small interfering RNA (siRNA) against C/EBPβ (C/EBPβ siRNA) (#1-3) were obtained, and a scrambled siRNA was used as the control (Con siRNA). PCR was performed to verify the knockdown efficiency of C/EBPP siRNA in HaCaT cells; C/EBPβ siRNA#1 was proved to be most effective at 120nM,48h, thus was selected for further research. Western bloting was performed to reveal that 120nM C/EBPβ siRNA, after 48h incubation, knockdown the C/EBPβ level of TNIP1 shRNA HaCaT cells to 60% of control (TNIP1 shRNA HaCaT+Con siRNA), and the knockdown of C/EBPβ resulted in downregulation of CK6 expression in TNIP1 shRNA HaCaT, which was decreased to 51% of TNIP1 shRNA HaCaT. Thus TNIP1 partially work through C/EBPβ to modulate the expression level of CK6. Notably, the Con siRNA we employed showed unspecific effects on C/EBPp level, as it depressed the C/EBPβ level of Con siRNA-treated TNIP1 shRNA HaCaT to 88% of that in TNIP1 shRNA HaCaT (p=0.41).20uM Erkl/2 inhibitor U0126 was used to incubate with TNIP1 shRNA HaCaT cells for 12h to inhibit p-Erkl/2 level, which was persistant upregulated in TNIP1 shRNA infected cells, and Dimethyl sulfoxide (DMSO) was used as the control. Then western bloting was carried out and demonstrated that U0126 incubation led to the p-Erkl/2 level of TNIP1 shRNA HaCaT decreased to 51% of control, and the CK6 level decreased to 47% of control when compared with DMSO treated TNIP1 shRNA HaCaT, but still higher than that in HaCaT. However, the inhibition of Erkl/2 led to the level of C/EBPβ increased by 160% when compared with DMSO treated cells. Further intensive cell signaling studies were needed to reveal how Erkl/2 negatively regulated the level of C/EBPβ. One possible reason might be the phosphorylation of Erkl/2 led to the phosphorylation of C/EBPβ, which was the unactive form of C/EBPβ, without transcriptional activity. It’s hard to clarify which part, downregulation of p-Erkl/2 or upregulation of C/EBPβ or even some unclear reason, to play crucial role in CK6 decreasement since blocking kinase Erkl/2 via the pharmacologic inhibitor U0126 might have a broad impact on cell signaling. However, it could be safely concluded that TNIP1 partially worked through Erkl/2 to modulate the synthesis of CK6 in HaCaT cells.Part IV A widely accepted IMQ-induced psoriasis-like mice model was used for study in vivo. In our research, the 8-10w, male BALB/c mice were randomly divided into 3 groups, one group received intradermal injection of TNIP1 shRNA lentivirus and topical IMQ treatment, one group were administrated with Con shRNA lentivirus injection and topical IMQ treatment, and the other group were given TNIP1 shRNA injection and control lotion treatment. The mice were shaved on back, intradermally injected 7.5×107TU lentiviral particles; 7d later the cream containing 5% IMQ or the control lotion was topically applied onto the back skin for consecutive 6 days to induce psoriatic lesion. And the clinical score of redness, scale and skin elevation was recorded daily after the startment of IMQ treatment. The score was stratified as follows:0 (negative),1 (weak),2 (moderate),3 (severe) and 4 (extremely severe), and the summary of these three scores was cumulative psoriasis score. Finally the mice were sacrificed and the skin around the injection area was collected for pathological examination with hematoxylin-eosin (HE) staining. As the clinical scores reflected, the IMQ treatment led to markedly increased redness, scaling, and skin elevation in TNIP1 shRNA mice compared with Con shRNA mice. Redness was the earlist symptom as it became obvious on TNIP1 shRNA mice skin after 2 days of IMQ treatment, while skin elevation was latest to be observed, after 4 days of IMQ treatment. And histological examination of skin sections from IMQ-treated TNIP1 shRNA mice showed more significant epidermal hyperplasia, more severe hyperkeratosis and parakeratosis, and more neutrophils infiltration, which implied the downregulated TNIP1 affected not only the proliferation but also the differentiation of epidermal cells. Notably the epidermis thickness of IMQ-treated TNIP1 shRNA mice was quite uneven, which might be due to the uneven distribution of lentiviral particles by intradermal injection. The control lotion treatment on TNIP1 shRNA mice led to no obvious clinical menifestation or pathological changes.Conclusion:1. The TNIPl gene level increased in the epidermis of psoriatic skin, while the TNIP1 protein level decreased when compared with normal control skin. The disregulation of TNIP1 may be involved in pathogenesis of psoriasis. TNIP1 was effectively downregulated both in vivo and in vitro by the lentiviral vector expressing TNIP1 shRNA, and was upregulated in vitro by the lentiviral vector expressing rTNIP1;2. TNIP1 was involved in the proliferation of human keratinocyte as confirmed by detection of CK6 expression level, CCK8 assay and BrdU assay. It promotes the cell growth in TNIP1 shRNA HaCaT/PHKs, while surpressed the cell proliferation in rTNIP1 HaCaT/PHKs;3. p-Erkl/2 and C/EBPβ level increased in TNIP1 shRNA HaCaT/PHKs while decreased in rTNIP1 HaCaT/PHKs. TNIP1 partially works through two important signaling pathways, Erk1/2 and C/EBPβ, to modulate the cell growth;4. Downregulated TNIP1 exaggerates psoriasis-like dermatitis in an IMQ-induced mouse model, thus TNIP1 may have a protective role in pathogenesis of psoriasis.