Claudin-1 Is a p63 Target Gene with a Crucial Role in Epithelial Development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias

Gender Differences Associated with the Prognostic Value of BPIFB4 in COVID-19 Patients: A Single-Center Preliminary Study

In the ongoing global COVID-19 pandemic, male sex is a risk factor for severe disease and death, and the reasons for these clinical discrepancies are largely unknown. The aim of this work is to study the influence of sex on the course of infection and the differences in prognostic markers between genders in COVID-19 patients. Our cohort consisted of 64 adult patients (n = 34 men and n = 30 women) with PCR-proven SARS-CoV-2 infection. Further, a group of patients was characterized by a different severity degree (n = 8 high- and n = 8 low-grade individuals for both male and female patients). As expected, the serum concentrations of LDH, fibrinogen, CRP, and leucocyte count in men were significantly higher than in females. When serum concentrations of the inflammatory cytokines, including IL-6, IL-2, IP-10 and IL-4 and chemokines like MCP-1, were measured with multiplex ELISA, no significant differences between male and female patients were found. In COVID-19 patients, we recently attributed a new prognostic value to BPIFB4, a natural defensin against dysregulation of the immune responses. Here, we clarify that BPIFB4 is inversely related to the disease degree in men but not in women. Indeed, higher levels of BPIFB4 characterized low-grade male patients compared to high-grade ones. On the contrary, no significant difference was reported between low-grade female patients and high-grade ones. In conclusion, the identification of BPIFB4 as a biomarker of mild/moderate disease and its sex-specific activity would open an interesting field for research to underpin gender-related susceptibility to the disease

DLX5, FGF8 and the Pin1 isomerase control ΔNp63α protein stability during limb development: a regulatory loop at the basis of the SHFM and EEC congenital malformations.

Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1-ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

The AEC mutations subvert p63 transcriptional potential.

<p>A) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for TAp63 mutants were cotransfected (0.025, 0.05, 0.1 µg) (striped, light grey and dark grey bars respectively). B) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for ΔNp63 mutants were cotransfected (0.025, 0.05, 0.1 µg) (striped, light grey and dark grey bars respectively). C) 0.05 µg of TAp63α or TA-AEC518 were transfected either alone or together in order to reproduce the heterozygous state found in AEC patients. D) 0.05 µg of ΔNp63α or ΔN-AEC518 were transfected either alone or together in order to reproduce the heterozygous state found in AEC patients. Cells were lysed after 24 hours and luciferase activity was determined. The basal activity of the reporter plasmids was set to 1. Data are presented as fold activation/repression relative to the sample without effectors. Each bar of the histogram represents the mean of three independent transfection duplicates. Standard deviations are indicated.</p

<i>Cldn-1</i> expression is down-regulated upon siRNA to <i>ΔNp63</i>.

<p>A) Primary mouse keratinocytes were transfected by control siRNA (siGFP), scrambled siRNA (Dharmacon) or <i>ΔNp63</i> specific siRNA (siΔNp63). 24 hours after transfection total RNA was extracted and subjected to RealTime PCR to verify <i>ΔNp63</i> and <i>p53</i> downregulation. Results are expressed as mean expression of three different samples. Standard Deviations (SD) are indicated. B) <i>Cldn-1</i>, <i>3</i> and <i>-10</i> expressions in the same samples as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002715#pone-0002715-g001" target="_blank">Figure 1A</a>. Expression of the three genes was normalized to <i>CyclophilinA</i> expression. SD from three different samples is indicated.</p

The <i>Cldn-1</i> promoter is regulated by the ΔNp63_ isoforms.

<p>A) Schematic representation of the <i>Cldn-1</i> promoter constructs used. White boxes: <i>Sp1</i> binding sites. Black boxes: <i>p53</i> binding sites. B) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for the p63 isoforms and p53 were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). C) U2OS cells were transfected with the deletion constructs of the <i>Cldn-1</i> reporter plasmid (0.3 µg). Different quantities of expression plasmids for the ΔNp63α were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). D) U2OS cells were transfected with the wild- type or the mutated −61 bp promoter constructs (0.3 µg). Different quantities of expression plasmids for the ΔNp63α were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). Cells were lysed after 24 hours and luciferase activity was determined. The basal activity of the reporter plasmids was set to 1. Data are presented as fold activation/repression relative to the sample without effectors. Each bar of the histogram represents the mean of three independent transfection duplicates. Standard deviations are indicated.</p

ΔΝp63_α functionally interacts <i>in vivo</i> with the <i>Cldn-1</i> gene.

<p>A) Schematic representation of the human, rat and mouse <i>Cldn-1</i> promoter regions. Two regions of high interspecies homology were identified (R1 and R2, black and striped boxes respectively). The degree of homology within R1 and R2 is shown on the right. R3, mapping at −3.5 Kb from the ATG of the <i>Cldn-1</i> gene, did not show any homology and was used as negative control. B) Three amplicons centered on R1, R2 and R3 of the <i>Cldn-1</i> gene were used in semiquantitative PCR amplifications with chromatin extracted from mouse primary keratinocytes, induced with Ca²⁺ for 24 hours, and immunoprecipitated with anti-p63 antibodies (4A4, Santa Cruz Biotech). R1 and R2 were positives with the anti-p63 antibodies while R3 was negative. As positive control, oligonucleotides annealing to the <i>IKKα</i> mouse promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002715#pone.0002715-Marinari1" target="_blank">[25]</a> were used. C) H1299 cells were treated with 20 µM Doxicycline in order to induce ΔNp63α expression. Thirty hours after induction mRNA was extracted from uninduced (-Dox) and induced (+Dox) cells and levels of endogenous <i>Cldn-1</i> and <i>GAPDH</i> were assessed by semiquantitative RT-PCR. D) Expression of the Cldn-1 protein is clearly detected upon ΔNp63α expression in H1299 cells. E) RNA extracted from mouse HL at E10.5, E11.5 and E12.5 (white, grey and black bars respectively) were used to verify <i>ΔNp63</i> and <i>Cldn-1</i> levels of expression.</p