Nicotinamide Prevents UVB- and Oxidative Stress‒Induced Photoaging in Human Primary Keratinocytes.

Nicotinamide (NAM), a NAM adenine dinucleotide precursor, is known for its benefits to skin health. Under standard culture conditions, NAM delays the differentiation and enhances the proliferation of human primary keratinocytes, leading to the maintenance of stem cells. In this study, we investigated the effects of NAM on photoaging in two-dimensional human primary keratinocyte cultures and three-dimensional organotypic epidermal models. In both models, we found that UVB irradiation and hydrogen peroxide induced human primary keratinocyte premature terminal differentiation and senescence. In three-dimensional organotypics, the phenotype was characterized by a thickening of the granular layer expressing filaggrin and loricrin, but thinning of the epidermis overall. NAM limited premature differentiation and ameliorated senescence, as evidenced by the maintenance of lamin B1 levels in both models, with decreased lipofuscin staining and reduced IL-6/IL-8 secretion in three-dimensional models, compared to those in UVB-only controls. In addition, DNA damage observed after irradiation was accompanied by a decline in energy metabolism, whereas both effects were partially prevented by NAM. Our data thus highlight the protective effects of NAM against photoaging and oxidative stress in the human epidermis and pinpoint DNA repair and energy metabolism as crucial underlying mechanisms.

Nicotinamide Metabolism Modulates the Proliferation/Differentiation Balance and Senescence of Human Primary Keratinocytes.

Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NAD+), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but the underlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show that NAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In 2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers and increases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared to controls. By contrast, preventing the conversion of NAM to NAD+ using FK866 leads to premature human primary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effects are rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NAD+ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NAD+ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.

Dual Role of the Anaphase Promoting Complex/Cyclosome in Regulating Stemness and Differentiation in Human Primary Keratinocytes.

Cdc20 and Cdh1 activate the anaphase-promoting complex/cyclosome, a master cell cycle regulator. Although cell cycle modifications occur during differentiation of stem cells, a role for the anaphase-promoting complex/cyclosome on stem cell fate has not been established in embryonic or adult human tissues. We found that differentiated human primary keratinocytes from the skin express extremely low levels of Cdc20 compared with human primary keratinocyte stem cells (holoclones). In agreement with this, staining of human skin biopsies showed that Cdc20 is expressed in occasional cells from the basal and epibasal layers of the epidermis and is absent from the differentiated layers. Conversely, Cdh1 is preferentially expressed in differentiated cells. Interestingly, partial silencing of Cdc20 enhanced differentiation, indicating that loss of Cdc20 might be a cause rather than a consequence of terminal differentiation. By contrast, Cdh1 silencing induced the opposite cellular phenotype, which was characterized by an increase in stemness, cellular proliferation, and loss of differentiation markers. These data pinpoint the anaphase-promoting complex/cyclosome as a key regulator of adult stem cell fate. They also demonstrate the critical and opposing roles of Cdc20 and Cdh1 in controlling the balance between human primary keratinocyte proliferation and differentiation, and therefore in regulating skin homeostasis.

Stranglehold on the spindle assembly checkpoint: the human papillomavirus E2 protein provokes BUBR1-dependent aneuploidy.

The Human Papillomavirus (HPV) E2 protein, which inhibits the E6 and E7 viral oncogenes, is believed to have anti-oncogenic properties. Here, we challenge this view and show that HPV-18 E2 over-activates the Spindle Assembly Checkpoint (SAC) and induces DNA breaks in mitosis followed by aneuploidy. This phenotype is associated with interaction of E2 with the Mitotic Checkpoint Complex (MCC) proteins Cdc20, MAD2 and BUBR1. While BUBR1 silencing rescues the mitotic phenotype induced by E2, p53 silencing or presence of E6/E7 (inactivating p53 and increasing BUBR1 levels respectively) both amplify it. This work pinpoints E2 as a key protein in the initiation of HPV-induced cervical cancer and identifies the SAC as a target for oncogenic pathogens. Moreover, our results suggest a role of p53 in regulating the mitotic process itself and highlight SAC over-activation in a p53-negative context as a highly pathogenic event.

Localization of HPV-18 E2 at mitochondrial membranes induces ROS release and modulates host cell metabolism.

Papillomavirus E2 proteins are predominantly retained in the nuclei of infected cells, but oncogenic (high-risk) HPV-18 and 16 E2 can shuttle between the host nucleus and cytoplasm. We show here that cytoplasmic HPV-18 E2 localizes to mitochondrial membranes, and independent mass spectrometry analyses of the E2 interactome revealed association to the inner mitochondrial membrane including components of the respiratory chain. Mitochondrial E2 association modifies the cristae morphology when analyzed by electron microscopy and increases production of mitochondrial ROS. This ROS release does not induce apoptosis, but instead correlates with stabilization of HIF-1α and increased glycolysis. These mitochondrial functions are not shared by the non-oncogenic (low-risk) HPV-6 E2 protein, suggesting that modification of cellular metabolism by high-risk HPV E2 proteins could play a role in carcinogenesis by inducing the Warburg effect.

HPV-18 E2

The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1^E4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2^E4 transcripts resulting from 2 splice donor sites in the 5' part of E2, while the splice acceptor site is the one used for E1^E4. Both E2^E4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2^E4-S and E2^E4-L. Whereas we could not differentiate E2^E4-S from E1^E4 in vivo, E2^E4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2^E4 products.