Role of FGFR2b expression and signaling in keratinocyte differentiation. Sequential involvement of PKCδ and PKCα

The tumor suppressor epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) induces human keratinocyte early differentiation. Moreover, protein kinases C (PKCs) are known to regulate the differentiation program in several cellular contexts, including keratinocytes. Therefore, in this paper we propose to clarify if FGFR2b could play a role also in the late steps of keratinocyte differentiation and to assess if this receptor-induced process would sequentially involve PKCδ and PKCα isoforms. Immunofluorescence, biochemical, and molecular approaches, performed on 2D cultures or 3D organotypic rafts of human keratinocytes overexpressing FGFR2b by stable transduction, showed that receptor signaling induced the precocious onset and an accelerated progression of keratinocyte differentiation, indicating that FGFR2b is a crucial regulator of the entire program of keratinocyte differentiation. In addition, the use of specific inhibitors and gene silencing approaches through specific siRNA demonstrated that PKCδ controls the onset of FGFR2b-triggered differentiation, while PKCα plays a role restricted to the terminal stages of the process. Molecular analysis revealed that the two PKC isoforms sequentially act via induction of KLF4 and DLX3, two transcription factors linked by negative loops to p63, suggesting that p63 would represent the hub molecule at the crossroad of an intricate signaling network downstream FGFR2b, involving multiple PKC-induced transcription factors

The aberrant expression of the mesenchymal variant of FGFR2 in the epithelial context inhibits autophagy

Signaling of the epithelial splice variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, epithelial mesenchymal transition (EMT) and tumorigenic features. Here we analyzed in the human keratinocyte cell line, as well as in primary cultured cells, the possible impact of FGFR2c forced expression on the autophagic process. Biochemical and quantitative immunofluorescence analysis, coupled to the use of autophagic flux sensors, specific substrate inhibitors or silencing approaches, showed that ectopic expression and the activation of FGFR2c inhibit the autophagosome formation and that AKT/MTOR is the downstream signaling mainly involved. Interestingly, the selective inhibition of AKT or MTOR substrates caused a reversion of the effects of FGFR2c on autophagy, which could also arise from the imbalance of the interplay between AKT/MTOR pathway and JNK1 signaling in favor of JNK1 activation, BCL-2 phosphorylation and possibly phagophore nucleation. Finally, silencing experiments of depletion of ESRP1, responsible for FGFR2 splicing and consequent FGFR2b expression, indicated that the switching from FGFR2b to FGFR2c isoform could represent the key event underlying the inhibition of the autophagic process in the epithelial context. Our results provide the first evidence of a negative impact of the out-of-context expression of FGFR2c on autophagy, suggesting a possible role of this receptor in the modulation of the recently proposed negative loop between autophagy and EMT during carcinogenesis

Interplay between FGFR2b-induced autophagy and phagocytosis: role of PLCγ-mediated signalling

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation

Autophagy in human keratinocytes: role of the fibroblast growth factor receptor 2b and its signaling

Signaling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. In this work I investigated, in our cell model of HaCaT keratinocytes, if the two processes might be related and the possible involvement of PLCγ signaling in the autophagy triggered by FGFR2b activation. Using fluorescence and electron microscopy we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signaling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signaling, which in turn activates JNK1 via PKCδ. Finally we found that, in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways depend on the level of receptor expression. These results suggest that FGFR2b signaling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation. Since the early oncoprotein E5 of the human papillomavirus type 16 (16E5) is able to down-regulate FGFR2b expression and since it has been recently proposed a possible role of the entire “early protein pool” of HPV16 in inhibiting the autophagic process in epithelial cells, I also proposed to investigate the possible impairment of FGFR2b-induced autophagy in keratinocytes expressing 16E5. The results showed that the presence of 16E5 strongly inhibited the autophagic process, while forced expression and activation of FGFR2b counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and FGFR2b might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation

Enzymatically Crosslinked Collagen as a Versatile Matrix for In Vitro and In Vivo Co‐Engineering of Blood and Lymphatic Vasculature

Adequate vascularization is required for the successful translation of many in vitro engineered tissues. This study presents a novel collagen derivative that harbors multiple recognition peptides for orthogonal enzymatic crosslinking based on sortase A (SrtA) and Factor XIII (FXIII). SrtA-mediated crosslinking enables the rapid co-engineering of human blood and lymphatic microcapillaries and mesoscale capillaries in bulk hydrogels. Whereas tuning of gel stiffness determines the extent of neovascularization, the relative number of blood and lymphatic capillaries recapitulates the ratio of blood and lymphatic endothelial cells originally seeded into the hydrogel. Bioengineered capillaries readily form luminal structures and exhibit typical maturation markers both in vitro and in vivo. The secondary crosslinking enzyme Factor XIII is used for in situ tethering of the VEGF mimetic QK peptide to collagen. This approach supports the formation of blood and lymphatic capillaries in the absence of exogenous VEGF. Orthogonal enzymatic crosslinking is further used to bioengineer hydrogels with spatially defined polymer compositions with pro- and anti-angiogenic properties. Finally, macroporous scaffolds based on secondary crosslinking of microgels enable vascularization independent from supporting fibroblasts. Overall, this work demonstrates for the first time the co-engineering of mature micro- and meso-sized blood and lymphatic capillaries using a highly versatile collagen derivative

CD146 expression profile in human skin and pre-vascularized dermo-epidermal skin substitutes in vivo

Background CD146 is a cell adhesion molecule whose expression profile in human skin has not yet been elucidated. Here, we characterize CD146 expression pattern in human skin, in particular in blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), which constitute human dermal microvascular endothelial cells (HDMECs), as well as in perivascular cells. Results We demonstrated that CD146 is a specific marker of BECs, but not of LECs. Moreover, we found CD146 expression also in human pericytes surrounding blood capillaries in human skin. In addition, we demonstrated that CD146 expression is up-regulated by the TNFα-IL-1β/NF-kB axis in both BECs and pericytes. Finally, we engineered 3D collagen hydrogels composed of HDMECs, CD146+ pericytes, and fibroblasts which developed, in vitro and in vivo, a complete microvasculature network composed of blood and lymphatic capillaries with pericytes investing blood capillaries. Conclusions Overall, our results proved that CD146 is a specific marker of BECs and pericytes, but not LECs in human skin. Further, the combination of CD146+ pericytes with HDMECs in skin substitutes allowed to bioengineer a comprehensive 3D in vitro and in vivo model of the human dermal microvasculature

Tagging the signatures of domestication in common bean (<i>Phaseolus vulgaris</i>) by means of pooled DNA samples

Background and Aims: The main aim of this study was to use an amplified fragment length polymorphism (AFLP)-based, large-scale screening of the whole genome of Phaseolus vulgaris to determine the effects of selection on the structure of the genetic diversity in wild and domesticated populations. Methods: Using pooled DNA samples, seven each of wild and domesticated populations of P. vulgaris were studied using 2506 AFLP markers (on average, one every 250 kb). About 10 % of the markers were also analysed on individual genotypes and were used to infer allelic frequencies empirically from bulk data. In both data sets, tests were made to determine the departure from neutral expectation for each marker using an FST-based method. Key Results: The most important outcome is that a large fraction of the genome of the common bean (16 %; P &lt;0·01) appears to have been subjected to effects of selection during domestication. Markers obtained in individual genotypes were also mapped and classified according to their proximities to known genes and quantitative trait loci (QTLs) of the domestication syndrome. Most of the markers that were found to be potentially under the effects of selection were located in the proximity of previously mapped genes and QTLs related to the domestication syndrome. Conclusions: Overall, the results indicate that in P. vulgaris a large portion of the genome appears to have been subjected to the effects of selection, probably because of linkage to the loci selected during domestication. As most of the markers that are under the effects of selection are linked to known loci related to the domestication syndrome, it is concluded that population genomics approaches are very efficient in detecting QTLs. A method based on bulk DNA samples is presented that is effective in pre-screening for a large number of markers to determine selection signatures

Relief food subsistence revealed by microparticle and proteomic analyses of dental calculus from victims of the Great Irish Famine

This study provides direct evidence of the dependency on relief food in Ireland around the time of the Great Famine (1845 to 1852) through dental calculus analysis of archaeological human remains. The findings show a dominance of corn (maize) and milk from the identified foodstuffs and corroborate the contemporaneous historical accounts of diet and subsistence. It shows that microparticle and proteomic analyses, even when based on small archaeological samples, can provide a valid snapshot of dietary patterns and food consumption. The occurrence of egg protein, generally only included in the diet for the better-off social classes, also highlights how these analytical techniques can provide unanticipated insights into the variability of diet in historical populations.Food and diet were class markers in 19th-century Ireland, which became evident as nearly 1 million people, primarily the poor and destitute, died as a consequence of the notorious Great Famine of 1845 to 1852. Famine took hold after a blight (Phytophthora infestans) destroyed virtually the only means of subsistence—}the potato crop{—}for a significant proportion of the population. This study seeks to elucidate the variability of diet in mid{–}19th-century Ireland through microparticle and proteomic analysis of human dental calculus samples (n = 42) from victims of the famine. The samples derive from remains of people who died between August 1847 and March 1851 while receiving poor relief as inmates in the union workhouse in the city of Kilkenny (52{°}39' N, -7{°}15' W). The results corroborate the historical accounts of food provisions before and during the famine, with evidence of corn (maize), potato, and cereal starch granules from the microparticle analysis and milk protein from the proteomic analysis. Unexpectedly, there is also evidence of egg protein{—}a food source generally reserved only for export and the better-off social classes{—which highlights the variability of the prefamine experience for those who died. Through historical contextualization, this study shows how the notoriously monotonous potato diet of the poor was opportunistically supplemented by other foodstuffs. While the Great Irish Famine was one of the worst subsistence crises in history, it was foremost a social disaster induced by the lack of access to food and not the lack of food availability

The incorporation of alpha-tocopherol and functional doses of phytosterol esters during cheesemaking does not affect DNA or mRNA dynamics of Streptococcus thermophilus and Lactococcus lactis throughout and after the end of ripening

Tocopherols and phytosterols are lipid-soluble molecules which have been widely used in the food industry. Nevertheless, the influence of these compounds on the performance of starter lactic acid bacteria (SLAB) in fermented foods has received little attention. Here, we assessed the behavior of Streptococcus thermophilus and Lactococcus lactis during the ripening of a functional Port Salut light cheese elaborated with these SLAB and with alpha-tocopherol and phytosterol esters as bioactive molecules. Functional and control cheeses were manufactured at an industrial plant and sampled at 7, 21, 40, 60 and 90 days after elaboration for real-time quantitative PCR (qPCR) or reverse transcription-qPCR (RT-qPCR) experiments. Target DNA and mRNA from both SLAB were detected after 90 days of elaboration in both functional and control cheeses, supporting their potential role in generating flavor metabolites. Furthermore, here we showed for the first time that the addition of alpha-tocopherol and functional doses of phytosterols did not affect DNA or mRNA dynamics of these SLAB during cheesemaking, throughout and after the end of ripening. Therefore, our results support the use of cheese manufactured with both S. thermophilus and L. lactis as an optimal delivery system for these beneficial bioactive compounds.Inst. de Tecnol. de los Alimentos- ITAFil: Pega, Juan Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA).LABINTEX. Centre de Coopération Internationale en Recherche Agronomique pour le Déeveloppement. Déepartement PERSYST, UMR Qualisud; FranciaFil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Diaz, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Ruzal, Sandra Monica Consejo Nacional de Investigaciones Científicas y Téecnica; ArgentinaFil: Perez, Carolina Daiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossetti, Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Rizzo, Sergio Anibal. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentin

Bioprinting and plastic compression of large pigmented and vascularized human dermo-epidermal skin substitutes by means of a new robotic platform

Extensive availability of engineered autologous dermo-epidermal skin substitutes (DESS) with functional and structural properties of normal human skin represents a goal for the treatment of large skin defects such as severe burns. Recently, a clinical phase I trial with this type of DESS was successfully completed, which included patients own keratinocytes and fibroblasts. Yet, two important features of natural skin were missing: pigmentation and vascularization. The first has important physiological and psychological implications for the patient, the second impacts survival and quality of the graft. Additionally, accurate reproduction of large amounts of patient’s skin in an automated way is essential for upscaling DESS production. Therefore, in the present study, we implemented a new robotic unit (called SkinFactory) for 3D bioprinting of pigmented and pre-vascularized DESS using normal human skin derived fibroblasts, blood- and lymphatic endothelial cells, keratinocytes, and melanocytes. We show the feasibility of our approach by demonstrating the viability of all the cells after printing in vitro, the integrity of the reconstituted capillary network in vivo after transplantation to immunodeficient rats and the anastomosis to the vascular plexus of the host. Our work has to be considered as a proof of concept in view of the implementation of an extended platform, which fully automatize the process of skin substitution: this would be a considerable improvement of the treatment of burn victims and patients with severe skin lesions based on patients own skin derived cells