Jian-Zhong Qin

Skin immune sentinels in health and disease

Human skin and its immune cells provide essential protection of the human body from injury and infection. Recent studies reinforce the importance of keratinocytes as sensors of danger through alert systems such as the inflammasome. In addition, newly identified CD103+ dendritic cells are strategically positioned for cross-presentation of skin-tropic pathogens and accumulating data highlight a key role of tissue-resident rather than circulating T cells in skin homeostasis and pathology. This Review focuses on recent progress in dissecting the functional role of skin immune cells in skin disease.

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-κB 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-κB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-κB, 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-κB but not early differentiation.

Caspase activation and disruption of mitochondrial membrane potential during UV radiation-induced apoptosis of human keratinocytes requires activation of protein kinase C.

The induction of apoptosis in human keratinocytes by UV radiation involves caspase-mediated cleavage and activation of protein kinase C delta (PKCδ). Here we examined the role of PKC activation in caspase activation and disruption of mitochondria function by UV radiation. Inhibition of PKC partially blocked UV radiation-induced cleavage of PKCδ, pro-caspase-3, and pro-caspase-8, and the activation of these caspases. PKC inhibition also blocked the UV-induced loss of mitochondria membrane potential, but did not block the release of cytochrome c from mitochondria. Expression of the active catalytic domain of PKCδ was sufficient to induce apoptosis and disrupt mitochondrial membrane potential, however a kinase inactive PKCδ catalytic domain did not. Furthermore, the PKCδ catalytic fragment generated following UV radiation localized to the mitochondria fraction, as did ectopically expressed PKCδ catalytic domain. These results identify a functional role for PKC activation in potentiating caspase activation and disrupting mitochondrial function during UV-induced apoptosis.

Cutting Edge: A Critical Functional Role for IL-23 in Psoriasis

Interleukin-23 is a key cytokine involved in the generation of Th17 effector cells. Clinical efficacy of an anti-p40 mAb blocking both IL-12 and IL-23 and disease association with single nucleotide polymorphisms in the IL23R gene raise the question of a functional role of IL-23 in psoriasis. In this study, we provide a comprehensive analysis of IL-23 and its receptor in psoriasis and demonstrate its functional importance in a disease-relevant model system. The expression of IL-23 and its receptor was increased in the tissues of patients with psoriasis. Injection of a mAb specifically neutralizing human IL-23 showed IL-23–dependent inhibition of psoriasis development comparable to the use of anti-TNF blockers in a clinically relevant xenotransplant mouse model of psoriasis. Together, our results identify a critical functional role for IL-23 in psoriasis and provide the rationale for new treatment strategies in chronic epithelial inflammatory disorders.

Role of NF-κB in the Apoptotic-resistant Phenotype of Keratinocytes

Several studies point to a role for NF-κB 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)-γ promotes increased epidermal thickness producing psoriatic lesions. While keratinocytes derived from psoriatic plaque resist apoptosis, and combination of TPA and IFN-γ activates NF-κB, the molecular mechanism linking NF-κB activation and keratinocyte apoptosis resistance was unknown. Therefore, we examined the ability of IFN-γ plus TPA to influence NF-κB 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-γ plus TPA produced a synergistic activation of NF-κB, compared with when each reagent was used individually. Normal keratinocytes when exposed to IFN-γ plus TPA acquired a resistance to UV light-induced apoptosis, which was dependent on NF-κB because expression of a dominant negative form of IκBα overcame the resistance. Compared with normal keratinocytes, HaCaT cells have a dysfunctional constitutive NF-κB signaling pathway not induced by IFN-γ and TPA, rendering HaCaT cells highly susceptible to UV-induced apoptosis. Thus, immortalized HaCaT cells have an abnormal constitutive and dysfunctional NF-κB signaling system. These results provide evidence that activation and proper regulation of NF-κB is essential for acquisition of an apoptotic-resistant phenotype for epidermal-derived keratinocytes.

Avoiding premature apoptosis of normal epidermal cells

DAVID LAWRENCE, ZAHRA SHAHROKH, SCOT MARSTERS, KIRSTEN ACHILLES, DANNY SHIH, BARBARA MOUNHO, KENNETH HILLAN, KLARA TOTPAL, LAURA DEFORGE, PETER SCHOW, JEFFREY HOOLEY, STEVE SHERWOOD, ROGER PAI, SUSAN LEUNG, LOLO KHAN, BRIAN GLINIAK, JEANINE BUSSIERE, CRAIG A. SMITH, STEPHEN S. STROM, SEAN KELLEY, JUDITH A. FOX, DEBORAH THOMAS & AVI ASHKENAZI Genentech Incorporated, South San Francisco, California, USA Immunex Corporation, Seattle, Washington, USA University of Pittsburgh, Pittsburgh, Pennsylvania, USA Email: aa@gene.com 1. Wiley, S.R. et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673–682 (1995). 2. Pitti, R.M. et al. Induction of apoptosis by Apo-2 Ligand, a new member of the tumor necrosis factor receptor family. J. Biol. Chem. 271, 12697–12690 (1996). 3. Ashkenazi, A. et al. Safety and anti-tumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104, 155–162 (1999). 4. Walczak, H. et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Med. 5, 157–163 (1999). 5. Gliniak, B. & Le, T. Tumor necrosis factor-related apoptosis-inducing ligand’s antitmor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res. 59, 6153–6158 (1999). 6. Roth, W. et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem. Biophys. Res. Commun. 265, 1999 (1999). 7. Chinnaiyan, A.M. et al. Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc. Natl. Acad. Sci. USA 97, 1754–1759 (2000). 8. Jo, M. et al. Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nature Med. 6, 564–567 (2000). 9. Nagata, S. Steering anti-cancer drugs away from the TRAIL. Nature Med. 6, 502–503 (2000). 10. Hymowitz, S.G. et al. Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Molec. Cell 4, 563–571 (1999). 11. Hymowitz, S.G. et al. A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry 39, 633–640 (2000). 12. Bodmer, J.-L., Meier, P., Tschopp, J. & Schneider, P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J. Biol. Chem. 275, 20632–20637 (2000). 13. Schneider, P. et al. Conversion of membranebound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205–1213 (1998). 14. Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F. & Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279 (1991). 15. Kischkel, F.C. et al. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611–620 (2000). 16. Ashkenazi, A. & Dixit, V.M. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255–260 (1999).

Enhanced Killing of Melanoma Cells by Simultaneously Targeting Mcl-1 and NOXA

By deciphering the dysregulation of apoptosis in melanoma cells, new treatment approaches exploiting aberrant control mechanisms regulating cell death can be envisioned. Among the Bcl-2 family, a BH3-only member, NOXA, functions in a specific mitochondrial-based cell death pathway when melanoma cells are exposed to a proteasome inhibitor (e.g., bortezomib). Some therapeutic agents, such as bortezomib, not only induce proapoptotic Bcl-2 family members and active conformational changes in Bak and Bax but also are associated with undesirable effects, including accumulation of antiapoptotic proteins, such as Mcl-1. To enhance the bortezomib-mediated killing of melanoma cells, the apoptotic pathway involving NOXA was further investigated, leading to identification of an important target (i.e., the labile Bcl-2 homologue Mcl-1 but not other survival proteins). To reduce Mcl-1 levels, melanoma cells were pretreated with several different agents, including Mcl-1 small interfering RNA (siRNA), UV light, or the purine nucleoside analogue fludarabine. By simultaneously triggering production of NOXA (using bortezomib) as well as reducing Mcl-1 levels (using siRNA, UV light, or fludarabine), significantly enhanced killing of melanoma cells was achieved. These results show binding interactions between distinct Bcl-2 family members, such as NOXA and Mcl-1, in melanoma cells, paving the way for novel and rational therapeutic combination strategies, which target guardians of the proapoptotic Bak- and Bax-mediated pathways, against this highly aggressive and often fatal malignancy.

Regulation of apoptosis by p53 in UV-irradiated human epidermis, psoriatic plaques and senescent keratinocytes.

The carcinogenic effects of sunlight in human epidermis may be thwarted by either: transient growth arrest and repair of DNA photodamage in keratinocytes (KCs); elimination of KCs with damaged DNA via apoptosis; or by stimulating a senescence switch whereby KCs become irreversibly growth arrested. Using normal human skin organ cultures and living epidermal equivalents, we demonstrate that in the proliferative basal layer, removal of KCs via apoptosis had a rapid onset (beginning within 2 h) following UV-light exposure generating progressively greater numbers of KCs with thymine dimers as the dose of UV-light was increased; involved induction of Apaf-1, activation of caspase-3, and was dependent on p53 activation as addition of a p53 chemical inhibitor blocked the apoptotic response. Suprabasal layer KCs underwent apoptosis at much later time points (>8 h). KCs in the basal layer repaired DNA damage more rapidly than KCs in suprabasal layers. Steady state levels of p53 increased in irradiated cells, and the increase was accompanied by phosphorylation of serine 9 and serine 15, but not serine 6 residues. By contrast, cultured KCs undergoing spontaneous replicative senescence were resistant to UV-induced apoptosis. Senescent KCs constitutively contained low levels of p53, which were neither increased nor phosphorylated or acetylated after UV-exposure and possessed minimal DNA binding activity, indicative of functional inactivation. Furthermore, treatment of senescent KCs with DNA damaging agent adriamycin did not result in activation of latent p53 or apoptosis. When KCs within psoriatic plaques were examined, they resembled senescent KCs in that they expressed p53, which was not phosphorylated or acetylated. Thus, UV-light induces DNA damage in human epidermal KCs triggering p53 activation, and subsequent apoptosis involving distinct cell layers and kinetics. However, the lack of p53 activation as seen in senescent KCs and psoriatic plaques, is associated with a relative resistance of KCs to UV-induced apoptosis. In conclusion, the sensitivity and resistance of KCs to apoptosis depends not only on the location within various layers of epidermis and levels of p53, but may also involve p53 activation via post-translational modifications.

Resistance to UV-induced apoptosis in human keratinocytes during accelerated senescence is associated with functional inactivation of p53

Compared to proliferating keratinocytes (KCs), growth‐arrested KCs are relatively resistant to UV‐light induced apoptosis. When KCs undergo confluency, or following exposure to anti‐proliferative agents such as IFN‐γ plus a phorbol ester–12‐O‐tetradecanoylyphorbol‐13‐acetate (TPA), they convert from a proliferative to a nonproliferative state resembling senescence. Since p53 regulates UV‐induced apoptosis of KCs, this report further characterizes p53 half‐life, post‐translational modifications, and transcriptional activity using cultured human KCs and living epidermal equivalents. The half‐life of p53 in KCs was longer than fibroblasts (greater than approximately 3 h vs. 30 min). Exposure of proliferating KCs to UV‐light induces post‐translational modifications of p53 including acetylation of lysine‐382 residues. By contrast, KCs undergoing irreversible growth arrest following confluency, or exposure to IFN‐γ plus TPA, were resistant to UV‐induced apoptosis, and failed to undergo the acetylation modification of p53. Exposure of KCs to IFN‐γ plus TPA reduced total cellular p53 levels and reduced the transcriptional activity of p53. Addition of Trichostatin A (TSA), an inhibitor of de‐acetylation, increased acetylation of lysine‐382 in confluent KCs, thereby enhancing susceptibility of confluent cultures to UV‐induced apoptosis. Pre‐treatment of epidermal equivalents with IFN‐γ plus TPA also blocked UV‐light induced increase in p53 levels, and reduced apoptosis. In conclusion, these studies demonstrate that growth arrested KCs may resist UV‐light induced apoptosis by inactivating the pro‐apoptotic function of p53. J. Cell. Physiol. 198: 100–109, 2004.

Abnormal NF-κB signaling pathway with enhanced susceptibility to apoptosis in immortalized keratinocytes

The transcriptional activation and proper regulation of NF-kappaB is known to be important to the apoptotic resistant phenotype of epidermal-derived keratinocytes. By comparing and contrasting the responses of normal foreskin-derived keratinocytes versus an immortalized skin-derived keratinocyte cell line (i.e. HaCaT cells), several molecular defects involving NF-kappaB signaling pathway were delineated in the immortalized keratinocytes. While exposure to IFN-gamma plus TPA produces growth arrest in both normal and immortalized keratinocytes, with rapid phosphorylation of MEKKI and recruitment of distinctive protein kinase C isoforms into the signalosome complex, subsequent molecular events necessary for NF-kappaB activation were abnormal in HaCaT cells. This disrupted NF-kappaB activation in HaCaT cells was accompanied by enhanced susceptibility to UV-light induced apoptosis, which was associated with elevated levels of E2F-1 and decreased TRAF1/TRAF2 levels. Additional defects in HaCaT cells included markedly diminished levels of IKKbeta (and lack of induction of kinase activity) in response to inflammatory stimuli, a failure of p21(WAF1/CIP1) to associate with CDK2, and a decreased association between p65 and p300. These studies suggest caution in using HaCaT cells as a substitute for normal keratinocytes to study apoptosis in the skin. Thus, it appears that while the immortalized cells can escape cell cycle checkpoints by elevated levels of E2F-1, an adverse biological consequence of such dysregulated cell cycle control is the inability to activate the anti-apoptotic NF-kappaB signaling pathway. Therefore, exploiting this apoptosis vulnerability in pre-malignant, or immortalized cells, prior to acquiring a death-defying phenotype characteristic of more advanced malignant cell types, provides the basis for an early interventional therapeutic strategy for cutaneous oncologists.