MiR-20a-3p regulates TGF-β1/Survivin pathway to affect keratinocytes proliferation and apoptosis by targeting SFMBT1 in vitro

Psoriasis is a common immune-mediated chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation, differentiation and apoptosis. However, the exact etiology and pathogenesis are still unclear. Evidence is rapidly accumulating for the role of microRNAs in psoriasis. It has been demonstrated that Interleukin-22 (IL-22) plays vital role in T cell-mediated immune response by interacting with keratinocytes in the pathogenesis of psoriasis. The aim of our study was to explore the possible functional role of miR-20a-3p in psoriasis and in IL-22 induced keratinocyte proliferation. Here, we found that miR-20a-3p was down-regulated in psoriatic lesions and in HaCaT cells (human keratinocyte cell line) treated by IL-22 stimulation. Functional experiments showed that overexpression of miR-20a-3p in HaCaT cells suppressed proliferation and induced apoptosis while its knockdown promoted cell proliferation and reduces cell apoptosis. Mechanistically, SFMBT1 was identified as the direct target of miR-20a-3p by dual luciferase reporter assay. SFMBT1 knockdown was demonstrated to inhibit cell growth and induced apoptosis, which was consistent with the function of miR-20a-3p upregulation in HaCaT cells. In addition, results of western blot analysis showed that miR-20a-3p upregulation or SFMBT1 knockdown changed the protein expression levels of TGF-β1 and survivin. Our findings suggest that miR-20a-3p play roles through targeting SFMBT1 and TGF-β1/Survivin pathway in HaCaT cells, and loss of miR-20a-3p in psoriasis may contribute to hyperproliferation and aberrant apoptosis of keratinocytes.     

A unified mitochondria mechanistic target of rapamycin acyl-coenzyme A dehydrogenase 10 signal relay modulation for metformin growth inhibition in human immortalized keratinocytes cells

Metformin exhibits antiproliferative and proapoptotic effects in a variety of diseases, characterized by malignant and nonmalignant hyperplastic cells; however, the underlying molecular mechanism of metformin in psoriasis has not been elucidated. In the current study, we found that after metformin treatment the proliferation of human immortalized keratinocytes (HaCaT) was significantly inhibited, while cell apoptosis was increased in a dose-dependent manner, accompanied with enhanced protein expression of acyl-coenzyme A dehydrogenase 10 (ACAD10). Furthermore, mechanism analysis revealed that ACAD10 expression is induced by downregulated activities of mechanistic target of rapamycin 1 (mTORC1) signaling rather than AMP-activated protein kinase signaling. The inactivation of mTORC1 by rapamycin pretreatment or rotenone-induced mitochondrial complex inhibition showed a similar effect because of the metformin treatment on the proliferation and apoptosis of HaCaT keratinocytes. Overexpression of mTORC1 almost reversed the antiproliferation and proapoptosis effects induced by metformin. This study showed that the metformin treatment inhibited HaCaT cells proliferation and promoted apoptosis by affecting the mitochondrial-mTORC1 signaling and elevated the ACAD10 expression. Hence, metformin can be used as a potential therapeutic agent for psoriasis.     

All-trans-retinoic acid and 13-cis-retinoic acid: pharmacokinetics and biological activity in different cell culture models of human keratinocytes.

Despite its known biological effect on epithelial cells, 13- CIS-retinoic acid shows low binding affinity to either cellular retinoic acid-binding proteins or nuclear retinoid receptors compared to its isomer all- TRANS-retinoic acid. We have postulated a prodrug-drug relation with 13- CIS-retinoic acid which isomerizes to all- TRANS-retinoic acid. On the other hand, the biological effects of these two compounds can differ in the widely used cell culture models of HaCaT and normal primary keratinocytes. In this study, we seeded HaCaT and normal keratinocytes at high densities leading to early confluence in order to imitate high keratinocyte proliferation, such as in acne and psoriasis, while to model decreased keratinocyte proliferation, as in aged and steroid-damaged skin, cells were seeded at a low density. High performance liquid chromatography was administered to examine retinoid uptake and metabolism in monolayer HaCaT and normal keratinocyte cultures and the 4-methylumbelliferyl heptanoate assay to estimate cell growth at different cell densities. Major qualitative and quantitative differences were detected in the two cell types regarding intracellular 13- CIS-retinoic acid isomerization to all- TRANS-retinoic acid. On the other hand, the two retinoic acid isomers showed similar effects on cell growth of both cell types tested with increasing proliferation at low cell densities, but being rather inactive at high ones in normal keratinocytes and exhibiting an antiproliferative effect in HaCaT keratinocytes. The missing effect of retinoids on cell proliferation in high seeding densities of normal keratinocytes may indicate that the normalizing activity of retinoids on hyperkeratotic diseases, such as acne or psoriasis, is likely to be carried out by modulation of cell differentiation than cell growth. On the other hand, induced keratinocyte proliferation in low seeding densities may provide an explanation for the acanthosis induced by topical retinoids in aged and steroid-damaged skin.     

Gardiquimod inhibits the expression of calcium-induced differentiation markers in HaCaT cells

Toll-like receptor 7 (TLR7) is an important member in pattern recognition receptors families. TLR7 signal pathway is involved in the physiological process in many type cells, but the impact of TRL7 on differentiation in the human keratinocytes is still unknown. In this study, we investigated the expression of TLR7 in keratinocytes, and the effect of TLR7 agonist gardiquimod on the expression of calcium (Ca²⁺)-induced keratinocytes differentiation markers in HaCaT cells. Immunohistochemistry and western-blotting analysis showed that TLR7 is expressed in basal keratinocytes of normal skin and in the human keratinocyte cell line HaCaT, but not expressed in the keratinocytes of psoriasis lesions. Pretreatment with gardiquimod could down-regulate Ca²⁺-induced differentiation marker expression and activate Raf-MEK-ERK and PI3K-AKT signal pathways in HaCaT cells. However, specific inhibitors studies showed that the down-regulation of the differentiation markers expression by gardiquimod was not dependent on the activation of these two pathways. TLR7 may play an important role in the pathogenesis of psoriasis through regulating the differentiation of the keratinocytes, and will give a new insight into the psoriasis.     

Ectopic expression of clusterin／apolipoprotein J or Bcl-2 decreases thesensitivity of HaCaT cells to toxic effects of ropivacaine

Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line,HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate—poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis,HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well established anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have potential utility in the local treatment of tumors.     

Participation of Gab1 and Gab2 in IL-22-mediated keratinocyte proliferation,migration, and differentiation

Interleukin-22 (IL-22) is one of the key mediators of keratinocyte alterations in psoriasis. IL-22 inhibits keratinocyte differentiation and induces the migration of human keratinocytes. Grb2-associated binder 1 (Gab1) has been shown to mediate epidermal growth factor-induced epidermal growth and differentiation via interaction with the Src homology-2-containing protein-tyrosine phosphatase (Shp2). In this investigation, we explore the role of Gab1 and Gab2 in IL-22-mediated keratinocyte activities. We show that both Gab1 and Gab2 were tyrosine phosphorylated in IL-22-stimulated HaCaT cells and human primary epidermal keratinocytes and contributed to the activation of Extracellular signal regulated kinase 1/2 (Erk1/2) through interaction with Shp2. We further demonstrate that HaCaT cells infected with adenoviruses expressing Shp2-binding-defective Gab1/2 mutants exhibited decreased cell proliferation and migration, as well as increased differentiation. Moreover, similar results were observed in HaCaT cells infected with adenovirus-based small interfering RNAs targeting Gab1 and/or Gab2. Altogether, these data underscore the critical roles of Gab1 and Gab2 in IL-22-mediated HaCaT cell proliferation, migration, and differentiation.     

Role of NF-kappaB in the apoptotic-resistant phenotype of keratinocytes

Several studies point to a role for NF-kappaB in modulating epidermal thickness and apoptotic susceptibility of keratinocytes. When phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) are topically applied, prominent epidermal thickening occurs, and exposure to interferon (IFN)-gamma promotes increased epidermal thickness producing psoriatic lesions. While keratinocytes derived from psoriatic plaque resist apoptosis, and combination of TPA and IFN-gamma activates NF-kappaB, the molecular mechanism linking NF-kappaB activation and keratinocyte apoptosis resistance was unknown. Therefore, we examined the ability of IFN-gamma plus TPA to influence NF-kappaB activity, gene expression, and response to UV light-induced apoptosis. These responses in normal keratinocytes were compared with immortalized keratinocytes (HaCaT cells). Exposure of normal keratinocytes to IFN-gamma plus TPA produced a synergistic activation of NF-kappaB, compared with when each reagent was used individually. Normal keratinocytes when exposed to IFN-gamma plus TPA acquired a resistance to UV light-induced apoptosis, which was dependent on NF-kappaB because expression of a dominant negative form of IkappaBalpha overcame the resistance. Compared with normal keratinocytes, HaCaT cells have a dysfunctional constitutive NF-kappaB signaling pathway not induced by IFN-gamma and TPA, rendering HaCaT cells highly susceptible to UV-induced apoptosis. Thus, immortalized HaCaT cells have an abnormal constitutive and dysfunctional NF-kappaB signaling system. These results provide evidence that activation and proper regulation of NF-kappaB is essential for acquisition of an apoptotic-resistant phenotype for epidermal-derived keratinocytes.     

Altered expression of prohibitin in psoriatic lesions and its cellular implication

Psoriasis is characterized by excessive proliferation of keratinocytes accompanying acanthosis and incomplete differentiation. Prohibitin was investigated by examining its function of HaCaT as well as psoriasis. Psoriatic involved skin revealed high level of prohibitin in the basal layer. Prohibitin was analyzed by applying RNAi (PHBi) with HaCaT, which demonstrated increased S-phase. PHBi showed enhanced sensitivity to anthralin-mediated cell death due to enhanced loss of mitochondrial membrane potential, suggesting a protective role of prohibitin against apoptosis. Collectively, prohibitin plays a role both in cell cycle regulation and in maintaining mitochondrial integrity, implying its association with pathogenesis of psoriasis.     

Chloramphenicol induces in vitro growth arrest and apoptosis of human keratinocytes

Chloramphenicol (CAP) is a broad-spectrum antibacterial drug that is widely used for topical application in ophthalmology and dermatology. In the present study we investigated the influence of CAP on human keratinocyte proliferation and apoptosis in vitro. CAP significantly inhibited proliferation and induced apoptosis of cultivated human keratinocytes, as revealed by incorporation of radioactive thymidine and flow cytometry analysis of intracellular esterase activity in fluorescein diacetate-stained cells, respectively. CAP-induced keratinocyte apoptosis was associated with activation of caspases and increased production of reactive oxygen species. The pro-apoptotic action of CAP was antagonized by the antioxidant agent N-acetylcysteine, the protein synthesis inhibitor cycloheximide, and PD98059, a selective inhibitor of extracellular signal-regulated kinase (ERK) activation. Taken together, these data indicate that CAP inhibits keratinocyte proliferation through induction of oxidative stress and ERK-mediated caspase-dependent apoptosis.     

A Mycobacterium ulcerans toxin,mycolactone, induces apoptosis in primary human keratinocytes and in HaCaT cells

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.     

Tumor Necrosis Factor-α-Induced Apoptosis in a Human Keratinocyte Cell Line (HaCaT) Is Counteracted by Transforming Growth Factor-α

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-α (TGF-α) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-α (TNF-α) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-α induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation (“DNA laddering”), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-α. The protective effect of TGF-α was abrogated by translation inhibition, indicating that it depended onde novoprotein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-α-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-α.     

Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier's disease

The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca2+ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2+/- mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca2+ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca2+ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca2+ and activation of nuclear factor-kappaB. Isotretinoin reduced Ca2+ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD.     

Evidence supporting a role for dihydroorotate dehydrogenase, bioenergetics, and p53 in selective teriflunomide-induced apoptosis in transformed versus normal human keratinocytes

We have demonstrated previously that the dihydroorotate dehydrogenase (DHODH) inhibitor teriflunomide (TFN) encourages apoptosis in transformed human keratinocytes. Here we sought to determine if this cytotoxic effect could be restricted to transformed keratinocytes relative to their normal human epidermal keratinocyte (NHEK) counterparts, and ascertain a potential mechanistic basis for the selectivity. The NHEK cells proliferated much slower than the premalignant HaCaT and malignant COLO 16 keratinocytes, and exogenous uridine added to the culture medium did not affect this growth. Similarly, DHODH expression and the bioenergetic characteristics of the normal cells were markedly dissimilar from those observed in the transformed cells indicating that de novo pyrimidine synthesis was involved with keratinocyte proliferation. Moreover, a short-term exposure to TFN caused a wild-type p53 response in the NHEK cells illustrating that pyrimidine metabolic stress could regulate this tumor suppressor protein in the normal cells. TFN-induced apoptosis occurred primarily in S phase HaCaT cells. This cell death was sensitive to uridine, an antioxidant, and a caspase inhibitor, and the suppression of Bcl-X_L and the induction of Mn superoxide dismutase preceded it. These events suggested that mitochondrial/redox stress was involved with the cytotoxic effect of TFN. Conversely, a long-term exposure to TFN caused G₀/G₁ arrest in the NHEK cells, which supported a cytoprotective role for p53 against TFN-induced apoptosis. Together, these results propose that TFN could be useful in the prevention or therapy of non-melanoma skin cancers and possibly other hyperproliferative keratinocytic diseases.     

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2

Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein-3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell-cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase-3 activation, Bcl-2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes.     

Apoptosis in proliferating, senescent, and immortalized keratinocytes.

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-kappaB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-kappaB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-kappaB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-kappaB but not early differentiation.     

n-3 and n-6 polyunsaturated fatty acids induce the expression of COX-2 via PPARgamma activation in human keratinocyte HaCaT cells

Polyunsaturated fatty acids (PUFA) n-3 inhibit inflammation, in vivo and in vitro in keratinocytes. We examined in HaCaT keratinocyte cell line whether eicosapentaenoic acid (EPA) a n-3 PUFA, gamma-linoleic acid (GLA) a n-6 PUFA, and arachidic acid a saturated fatty acid, modulate expression of cyclooxygenase-2 (COX-2), an enzyme pivotal to skin inflammation and reparation. We demonstrate that only treatment of HaCaT with GLA and EPA or a PPARgamma ligand (roziglitazone), induced COX-2 expression (protein and mRNA). Moreover stimulation of COX-2 promoter activity was increased by those PUFAs or rosiglitazone. The inhibitory effects of GW9662 and T0070907 (PPARgamma antagonists), on COX-2 expression and on stimulation of COX-2 promoter activity by EPA and GLA suggest that PPARgamma is implicated in COX-2 induction. Finally, PLA2 inhibitor methyl arachidonyl fluorophosphonate blocked the PUFA effects on COX-2 induction, promoter activity and arachidonic acid mobilization suggesting involvement of AA metabolites in PPAR activation. These findings demonstrate that n-3 and n-6 PUFA increased PPARgamma activity is necessary for the COX-2 induction in HaCaT human keratinocyte cells. Given the anti-inflammatory properties of EPA, we suggest that induction of COX-2 in keratinocytes may be important in the anti-inflammatory and protective mechanism of action of PUFAs n-3 or n-6.