RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development.

BACKGROUND: Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes. RESULTS: Cultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell-cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient's keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control. CONCLUSIONS: Altogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

RSPO1-mutated fibroblasts from non-tumoural areas of palmoplantar keratoderma display a cancer-associated phenotype.

R-spondin (RSPO)1 is a fibroblast-secreted protein that belongs to the R-spondin protein family which is essential for reproductive organ development, epithelial stem cell renewal and cancer induction or suppression. RSPO1 gene mutations cause palmoplantar hyperkeratosis with squamous cell carcinoma (SCC) of the skin, 46XX sex reversal and true hermaphroditism. To characterize RSPO1-deficient skin fibroblasts derived from two patients with mutations in RSPO1, with palmoplantar hyperkeratosis, recurrent SCC and 46XX sex reversal, to provide further insight into disease-related skin tumourigenesis. Fibroblast cultures from non-tumoural palmoplantar skin biopsies were established to evaluate features and properties that may be altered at cancer onset, i.e. proliferation, extracellular matrix contraction and invasion, as well as TGF-ß and matrix metalloproteinase (MMP) secretion. Fibroblasts demonstrated increased proliferative potential in vitro, a high level of collagen contraction and invasion by SCC cells, release of high levels of pro-inflammatory and pro-fibrotic TGF-ß, and increased expression of MMP1 and MMP3. Analysis of the expression of selected proteins associated with RSPO1-activated pathways confirmed sustained activation of the TGF-ß signalling pathway and indicated a loss of TGF-ß inhibitory feedback. Also, treatment of fibroblasts with a recombinant RSPO1 protein aggravated this pro-inflammatory phenotype, suggesting caution in designing therapeutic strategies based on restoration of protein function. Our findings indicate that fibroblasts from RSPO1-mutated patients behave similarly to cancer-associated fibroblasts. Chronic inflammation and fibrotic changes in palmoplantar skin may play a role in SCC development and recurrence, possibly by irreversibly activating the tumourigenic phenotype of fibroblasts.

The Secretome of Aged Fibroblasts Promotes EMT-Like Phenotype in Primary Keratinocytes from Elderly Donors through BDNF-TrkB Axis.

Age-related changes in the dermis can play a primary role in tumor initiation promoting the unrestrained proliferation of precancerous keratinocytes (KCs) through cytokines and GF secretion. We found a high percentage of epithelial-to-mesenchymal transition-like colonies raising in primary human KC cultures from old subjects after treatment with aged fibroblast supernatants (SPNs). Continuous extracellular signals were required for maintaining these changes. Conversely, the secretome did not induce epithelial-to-mesenchymal transition-like colonies in KCs from young subjects. SPN-treated aged KCs displayed the activation of pathways involved in the disjunction of cell‒cell adhesion, extracellular matrix remodeling, manifestation of a mesenchymal phenotype, and dedifferentiation programs. Moreover, they recovered proliferation and clonogenic ability and showed enhanced migration. We identified an age-related increase of the BDNF secretion from fibroblasts as well as of the expression of its receptor TrkB in KCs. BDNF treatment of aged KCs induced TrkB phosphorylation and recapitulated the modifications promoted by aged fibroblast SPN. Furthermore, the treatment with a specific antibody against BDNF or a TrkB antagonist inhibited the paracrine signaling preventing SPN-mediated morphological and molecular changes. Finally, BDNF induced signs of matrix invasion in a three-dimensional organotypic model. Therefore, we demonstrate that aged fibroblast SPN promotes phenotypic plasticity in KCs from the elderly through BDNF-TrkB axis.

Measles skin rash: Infection of lymphoid and myeloid cells in the dermis precedes viral dissemination to the epidermis.

Measles is characterized by fever and a maculopapular skin rash, which is accompanied by immune clearance of measles virus (MV)-infected cells. Histopathological analyses of skin biopsies from humans and non-human primates (NHPs) with measles rash have identified MV-infected keratinocytes and mononuclear cells in the epidermis, around hair follicles and near sebaceous glands. Here, we address the pathogenesis of measles skin rash by combining data from experimentally infected NHPs, ex vivo infection of human skin sheets and in vitro infection of primary human keratinocytes. Analysis of NHP skin samples collected at different time points following MV inoculation demonstrated that infection in the skin precedes onset of rash by several days. MV infection was detected in lymphoid and myeloid cells in the dermis before dissemination to the epidermal leukocytes and keratinocytes. These data were in good concordance with ex vivo MV infections of human skin sheets, in which dermal cells were more targeted than the epidermal cells. To address viral dissemination to the epidermis and to determine whether the dissemination is receptor-dependent, we performed experimental infections of primary keratinocytes collected from healthy donors. These experiments demonstrated that MV infection of keratinocytes is mainly nectin-4-dependent, and differentiated keratinocytes, which express higher levels of nectin-4, are more susceptible to MV infection than proliferating keratinocytes. Based on these data, we propose a model to explain measles skin rash: migrating MV-infected lymphocytes initiate the infection of dermal skin-resident CD150+ immune cells. The infection is subsequently disseminated from the dermal papillae to nectin-4+ keratinocytes in the basal epidermis. Lateral spread of MV infection is observed in the superficial epidermis, most likely due to the higher level of nectin-4 expression on differentiated keratinocytes. Finally, MV-infected cells are cleared by infiltrating immune cells, causing hyperemia and edema, which give the appearance of morbilliform skin rash.

Cockayne Syndrome Type A Protein Protects Primary Human Keratinocytes from Senescence.

Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype.

Recessive mutations in the neuronal isoforms of DST, encoding dystonin, lead to abnormal actin cytoskeleton organization and HSAN type VI.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.

Oxidative Stress-Induced miR-200c Disrupts the Regulatory Loop Among SIRT1, FOXO1, and eNOS.

AIMS: Reactive oxygen species (ROS) play a pivotal role in different pathologic conditions, including ischemia, diabetes, and aging. We previously showed that ROS enhance miR-200c expression, causing endothelial cell (EC) apoptosis and senescence. Herein, we dissect the interaction among miR-200c and three strictly related proteins that modulate EC function and ROS production: sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS), and forkhead box O1 (FOXO1). Moreover, the role of miR-200c on ROS modulation was also investigated. RESULTS: We demonstrated that miR-200c directly targets SIRT1, eNOS, and FOXO1; via this mechanism, miR-200c decreased NO and increased the acetylation of SIRT1 targets, that is, FOXO1 and p53. FOXO1 acetylation inhibited its transcriptional activity on target genes, that is, SIRT1 and the ROS scavengers, catalase and manganese superoxide dismutase. In keeping, miR-200c increased ROS production and induced p66Shc protein phosphorylation in Ser-36; this mechanism upregulated ROS and inhibited FOXO1 transcription, reinforcing this molecular circuitry. These in vitro results were validated in three in vivo models of oxidative stress, that is, human skin fibroblasts from old donors, femoral arteries from old mice, and a murine model of hindlimb ischemia. In all cases, miR-200c was higher versus control and its targets, that is, SIRT1, eNOS, and FOXO1, were downmodulated. In the mouse hindlimb ischemia model, anti-miR-200c treatment rescued these targets and improved limb perfusion. Innovation and Conclusion: miR-200c disrupts SIRT1/FOXO1/eNOS regulatory loop. This event promotes ROS production and decreases NO, contributing to endothelial dysfunction under conditions of increased oxidative stress such as aging and ischemia. Antioxid. Redox Signal. 27, 328-344.

Magnetic fields promote a pro-survival non-capacitative Ca2+ entry via phospholipase C signaling.

The ability of magnetic fields (MFs) to promote/increase Ca(2+) influx into cells is widely recognized, but the underlying mechanisms remain obscure. Here we analyze how static MFs of 6 mT modulates thapsigargin-induced Ca(2+) movements in non-excitable U937 monocytes, and how this relates to the anti-apoptotic effect of MFs. Magnetic fields do not affect thapsigargin-induced Ca(2+) mobilization from endoplasmic reticulum, but significantly increase the resulting Ca(2+) influx; this increase requires intracellular signal transduction actors including G protein, phospholipase C, diacylglycerol lipase and nitric oxide synthase, and behaves as a non-capacitative Ca(2+) entry (NCCE), a type of influx with an inherent signaling function, rather than a capacitative Ca(2+) entry (CCE). All treatments abrogating the extra Ca(2+) influx also abrogate the anti-apoptotic effect of MFs, demonstrating that MF-induced NCCE elicits an anti-apoptotic survival pathway.

Bmi-1 reduction plays a key role in physiological and premature aging of primary human keratinocytes.

Accumulation of senescent cells contributes to the reduced regenerative capacity in aged tissues. By evaluating the molecular pathways of senescence in relation to proliferative potential of primary keratinocyte cultures from young and old healthy donors, and from young patients with inherited defects leading to premature aging, we demonstrated that p16(INK4a) is a reliable marker of both physiological and premature epidermal aging. Analysis of the expression and activity of p16(INK4a) regulators showed that stem cell depletion, reduced proliferation, and p16(INK4a) upregulation in keratinocytes derived from the chronologically and prematurely aged epidermis strongly correlate with Bmi-1 downregulation. In highly proliferative tissues, replicative and premature senescence participate in determining senescent cell accumulation. Our findings demonstrated that Bmi-1 is downregulated in human keratinocytes during both in vitro processes, in parallel with p16(INK4a) upregulation and accomplishment of clonal conversion. When premature senescence was induced by specific exogenous stimuli, concomitant Ets-1 upregulation was also observed. Moreover, Bmi-1 inhibited Ets-1-mediated p16(INK4a) upregulation. Finally, Bmi-1 overexpression reduced p16(INK4a) promoter activity and decreased protein expression in aged and diseased keratinocytes, inducing a delay of clonal conversion and an increase of cell clonogenic ability. Altogether these findings underline a key role of Bmi-1 downregulation in enforcing aging in primary human keratinocytes.