Laura Leone

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways.

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high‐affinity‐specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin‐coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c‐Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: A new molecular mechanism to dampen mast cell function

Ligation of the high-affinity receptor for IgE (FcεRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, FcεRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of FcεRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating FcεRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the FcεRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of FcεRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.

Identification and Characterization of the Product Encoded by ORF69 of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT We report the identification and characterization of p33, the product of Kaposis sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco‐2 cells upon confluence‐induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up‐modulated in post‐confluent differentiated cultures compared with the pre‐confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up‐regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF‐10, whereas they were not stimulated by the EGFR ligands TGFα and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post‐confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up‐modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

Endocytic pathways and biological effects induced by UVB-dependent or ligand-dependent activation of the keratinocyte growth factor receptor

UVB exposure of epidermal cells is known to trigger early and late molecular pathways dependent on receptor tyrosine kinases and reactive oxygen species (ROS). We have recently reported that UVB irradiation induces tyrosine phosphorylation, kinase activation, and internalization of the receptor for the keratinocyte growth factor (KGFR), a paracrine mediator of epithelial growth, differentiation, and survival. Here we analyzed in more detail the UVB‐induced endocytic pathway of KGFR and the role of KGFR activation and internalization in regulating UVB‐promoted apoptosis and cell cycle arrest. Immunogold electron microscopy and confocal analysis revealed that the UVB‐induced endocytosis of KGFR occurs through clathrin‐coated pits and that the internalized receptors are sorted to the degradative route and reach the lysosomal compartment with a timing similar to that induced by their ligand KGF. Treatment with the anti‐oxidant Nacetylcysteine inhibited KGFR endocytosis, suggesting that the receptor internalization is mediated by the intracellular production of ROS. The ligand‐independent KGFR endocytic pathway induced by UVB requires receptor kinase activity and tyrosine phosphorylation and involves transient receptor ubiquitination. Inhibition of KGFR activity reduces both the KGF‐mediated proliferative response and the UVB‐promoted apoptotic cell death, indicating a different effect of ligand‐induced and UVB‐induced KGFR triggering. In addition, receptor internalization leads to protection from apoptosis caused by UVB exposure. Finally, we compared directly the behavior of KGFR with that of the epidermal growth factor receptor (EGFR) upon UVB exposure. Surprisingly, biochemical and immunofluorescence analysis showed that EGFR, differently from KGFR, does not undergo UVB‐induced tyrosine phosphorylation and internalization. Taken together, our results suggest a differential role of KGFR and EGFR in the response of epidermal cells to UVB possibly because KGFR endocytosis could be crucial for attenuation of survival signals in the suprabasal layers of human skin.

Expression and signaling of the tyrosine kinase FGFR2b/KGFR regulates phagocytosis and melanosome uptake in human keratinocytes

Membrane and actin cytoskeleton dynamics during phagocytosis can be triggered and amplified by the signal transduction of receptor tyrosine kinases. The epidermal keratinocytes appear to use the phagocytic mechanism of uptake to ingest melano‐somes released by the melanocytes and play a pivotal role in the transfer process. We have previously demonstrated that the keratinocyte growth factor KGF/ FGF7 promotes the melanosome uptake through activation of its receptor tyrosine kinase FGFR2b/KGFR. The aim of the present study was to investigate the contribution of KGFR expression, activation, and signaling in regulating the phagocytic process and the melanosome transfer. Phagocytosis was analyzed in vitro using fluorescent latex beads on human keratin‐ocytes induced to differentiate. Melanosome transfer was investigated in keratinocyte‐melanocyte cocul‐tures. KGFR depletion by small interfering RNA microinjection and overexpression by transfection of wild type or defective mutant KGFR were performed to demonstrate the direct effect of the receptor on phagocytosis and melanosome transfer. Colocalization of the phagocytosed beads with the internalized receptors in phagolysosomes was analyzed by optical sectioning and 3‐dimensional reconstruction. KGFR ligands triggered phagocytosis and melanosome transfer in differentiated keratinocytes, and receptor kinase activity and signaling were required for these effects, suggesting that FGFR2b/KGFR expression/activity and PLCγ signaling pathway play crucial roles in phagocytosis.—Belleudi, F., Purpura, V., Scrofani, C., Persechino, F., Leone, L., and Torrisi, M. R. Expression and signaling of the tyrosine kinase FGFR2b/KGFR regulates phagocytosis and melano‐some uptake in human keratinocytes. FASEB J. 25, 170–181 (2011). www.fasebj.org

Decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fibrosis fibroblasts

Autophagy is the main cellular pathway for degradation of long‐lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl‐2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down‐regulated in comparison with fibroblasts from normal lungs while the anti‐apoptotic protein Bcl‐2 expression was over‐expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl‐2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl‐2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction. J. Cell. Physiol. 228: 1516–1524, 2013.

Preliminary evidences on mitochondrial injury and impaired oxidative metabolism in breast cancer

Mitochondriopathy is emerging as a new cancer theory; however, the relevance of mitochondrial pathobiology in breast cancer has not yet been completely explored. Herein we report on altered expression levels of the oxidative phosphorylation system (OXPHOS) subunits, mitochondrial structural injury and impaired ATP content from a breast-infiltrating ductal carcinoma (IDC). With this purpose, a human mammary carcinoma (HMC-1) cell, referred to a human mammary epithelial cell (HMEC) line, was assayed for: a) OXPHOS levels by quantitative cryo-immunoelectron microscopy (CIEM) labeling; b) morphological characterization by a newly introduced damage grading (scale Mt-g1-3), calculated on the % of intact cristae carrying mitochondria; c) bioenergetic impairment by luminometric determinations of cellular ATP content and cytochemical visualization of COX activity. Drastic OXPHOS reduction was observed in HMC-1 cells for the succinate-dehydrogenase complex II SDH-B protein, while decreasing was reported for the NADH-ubiquinone oxidoreductase complex I NDUFS3 and the ubiquinol cytochrome c reductase complex III UQCRC2 subunits. A significant dropping was detected for the ATP-synthase complex V F1β protein. For the COX complex near-depletion of the mitochondrial-encoded COXI and no apparent variation of the COXIV subunits were observed. Injury grading was categorized assigning three levels of morphological damage in HMC-1 mitochondria: i) severe (4.6%), ii) moderate (23.1%), iii) slight (44.6%), corresponding to 0%, 1-50% and 51-75% of area occupied by intact cristae. ATP generation and COX activity appeared significantly reduced in HMC-1 cells. The structural damage grading here described could provide new insight on IDC mitochondrial impairment and represent hallmark in the breast cancer mitochondriopathy.

Extracorporeal Shock Wave Treatment (ESWT) Improves In Vitro Functional Activities of Ruptured Human Tendon-Derived Tenocytes

In vitro models of human tenocytes derived from healthy as well as from ruptured tendons were established, characterized and used at very early passage (P1) to evaluate the effects of Extracorporeal Shock Wave Treatment (ESWT). The molecular analysis of traditional tenocytic markers, including Scleraxis (Scx), Tenomodulin (Tnm), Tenascin-C (Tn-C) and Type I and III Collagens (Col I and Col III), permitted us to detect in our samples the simultaneous expression of all these genes and allowed us to compare their levels of expression in relationship to the source of the cells and treatments. In untreated conditions, higher molecular levels of Scx and Col I in tenocytes from pathological compared to healthy samples have been detected, suggesting – in the cells from injured tendon – the natural trigger of an early differentiation and repairing program, which depends by Scx and requires an increase in collagen expression. When ESWT (at the dose of 0.14 mJ/mm2) was applied to cultured tenocytes explanted from injured source, Scx and Col I were significantly diminished compared to healthy counterpart, indicating that such natural trigger maybe delayed by the treatment, in order to promote cellular repair. Herein, we show for the first time that ESWT enhances in vitro functional activities of ruptured tendon-derived tenocytes, such as proliferation and migration, which could probably contributes to tendon healing in vivo.

Cholesteatoma-associated fibroblasts modulate epithelial growth and differentiation through KGF/FGF7 secretion

The keratinocyte growth factor (KGF/FGF7), produced by stromal cells, is a key paracrine mediator of epithelial proliferation, differentiation and migration. Expression of the growth factor is increased in wound healing and in hyperproliferative epithelial diseases, as a consequence of the activation of dermal fibroblasts by the inflammatory microenvironment. The middle ear cholesteatoma, an aural epidermal pathology characterized by keratinocyte hyperproliferation and chronic inflammation, may represent a model condition to study the epithelial-mesenchymal interactions. To develop an in vitro model for this disease, we isolated and characterized human primary cultures of fibroblasts associated with the cholesteatoma lesion, analyzing their secretory behaviour and degree of differentiation or activation. Compared to the perilesional or control normal fibroblasts, all cultures derived from cholesteatoma tissues were less proliferating and more differentiated and their highly variable activated phenotype correlated with the secretion of KGF as well as of metalloproteases 2 and 9. Culture supernatants collected from the cholesteatoma-associated fibroblasts were able to increase the proliferation and differentiation of human keratinocytes assessed by the expression of Ki67 and keratin-1 markers. The single crucial contribution of the KGF released by fibroblasts on the keratinocyte biological response was shown by the specific, although partial, block induced by inhibiting the KGF receptor or by immunoneutralizing the growth factor. Altogether, these results suggest that the activation of the stromal fibroblasts present in the pathological tissue, and the consequent increased secretion of KGF, play a crucial role in the deregulation of the epidermal proliferation and differentiation.