Mauro Patrone

Hmgb1 Promotes Wound Healing of 3T3 Mouse Fibroblasts via Rage-Dependent ERK1/2 Activation

HMGb1 is a nuclear protein playing a role in DNA architecture and transcription. This protein has also been shown to function as a cytokine and to stimulate keratinocyte scratch wound healing. Due to the importance of finding new wound healing molecules, we have studied the effects of HMGb1 on fibroblasts, another major skin cell type, using the NIH 3T3 line. HMGb1 expression in these cells was assessed by Western blot, while its nuclear localization was pointed out by confocal immunofluorescence. HMGb1-induced cell proliferation with a maximum at a concentration of 10 nM, and such a dose also stimulated cell migration and scratch wound healing. Western blot analysis showed that HMGb1 activates ERK1/2, while the use of an anti-RAGE receptor-blocking antibody and of the selective MEK1/2 inhibitor PD98059 blocked ERK1/2 activation and wound healing responses to HMGb1. Taken together data show that HMGb1 promotes 3T3 fibroblast wound healing by inducing cell proliferation and migration, and that this occurs through the activation of the RAGE/MEK/ERK pathway. In conclusion, HMGb1 seems a good candidate for the development of medical treatments to be used on chronic or severe wounds.

HMGb1 promotes scratch wound closure of HaCaT keratinocytes via ERK1/2 activation

HMGb1 is a DNA-binding protein whose role as an extracellular cytokine in inflammation and tissue regeneration has also been reported. Given the importance of keratinocytes in wound healing, we have studied the mechanism of action of HMGb1 on HaCaT keratinocytes during in vitro scratch wound repair. Western blot and confocal immunofluorescence microscopy showed that these cells express significant amounts of HMGb1, that the protein is prevalently localized in the nucleus, and that its release by cells is negligible. Western blot also showed that these cells express the HMGb1 receptor RAGE. Cell exposure to HMGb1 in the absence of serum resulted in a stimulation of cell proliferation and ERK1/2 activation. HMGb1 also accelerated the wound closure of scratch wounded cells and promoted cell migration, as evaluated by a transwell assay. The HMGb1-induced increases of cell proliferation, cell migration, and wound closure were abolished by the MEK inhibitor PD98059. Taken together, data show that, although HMGb1 is not released by HaCaT, when applied exogenously it can induce a marked increase of the wound repair of these cells. Data also suggest that HMGb1 acts via the RAGE/MEK/ERK pathway. These results bring scientific support to the potential application of HMGb1 in regenerative medicine.

Secretion and binding of HMG1 protein to the external surface of the membrane are required for murine erythroleukemia cell differentiation

We show here that murine erythroleukemia (MEL) cells, following induction with hexamethylene bisacetamide, accumulate high mobility group (HMG)1 protein onto the external surface of the cell in a membrane‐associated form detectable by immunostaining with a specific anti‐HMG1 protein antibody. This association is maximal at a time corresponding to cell commitment. At longer times, immunostainable cells are progressively reduced and become almost completely undetectable along with the appearance of hemoglobin molecules. Binding to MEL cells does not affect the native molecular structure of HMG1 protein. The type of functional correlation between HMG1 protein and MEL cell differentiation is suggested by the observation that if an anti‐HMG1 protein antibody is added at the same time of the inducer almost complete inhibition of cell differentiation is observed, whereas if the antibody is added within the time period in which cells undergo through irreversible commitment, inhibition progressively disappears. A correlation between MEL cell commitment and the biological effect of HMG1 protein can thus be consistently suggested.

Platelet lysate stimulates wound repair of HaCaT keratinocytes

Background  Platelets play a pivotal role in wound healing. Their beneficial effect is attributed to the release of bioactive substances, although the involved mechanisms are mostly unknown.

Protein kinase C mRNA levels and activity in reconstituted normal human epidermis: Relationships to cell differentiation

Although keratinocytes are a major target of phorbol ester actions, the activity and the expression of the eight cloned protein kinase C (PKC) isoenzymes have not been studied in detail in human epidermis. Starting from normal human keratinocytes, we reconstituted in culture a multilayered epithelial tissue which presents many hystological, biochemical, and molecular features of the authentic epidermis and we used it as a model to investigate the PKC activity and mRNA levels. We found that i) PKC activity is higher in differentiated than in non-differentiated cells; ii) the mRNA levels of PKC delta and -eta/L, while are differently affected by spontaneous keratinocyte differentiation, are down-regulated during phorbol esters-induced cell differentiation. Our findings could represent a basis to investigate the involvement of PKC isoforms in the keratinocyte differentiation process.

Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients.

In neutrophils of patients with essential hypertension the NADPH-dependent O2- production elicited by stimulation with f-Met-Leu-Phe is three to four fold higher in comparison with neutrophils of normotensive control subjects. Neutrophils from hypertensive patients are less responsive to priming, by non-stimulating doses of the agonist, as compared to control cells, which following this pretreatment augment superoxide anion production up to levels close to those expressed by neutrophils from hypertensive patients. No difference in NADPH oxidase activity, between neutrophils from the two groups of subjects, was observed when the rate of O2- production was evaluated in a reconstructed cell-free system containing the membrane fraction and the cytosolic cofactors. These results are consistent with the hypothesis that differences in the functional organization of the oxidase at the membrane level in neutrophils of hypertensive are responsible for the enhanced O2- production following agonist stimulation.

Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKT

There is a growing interest for the clinical use of platelet derivates in wound dressing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances, though mechanisms are mostly unknown. We studied wound‐healing processes of human primary fibroblasts, by exposing cells to a platelet lysate (PL) obtained from blood samples. Crystal violet and tetrazolium salt (MTS) assays showed dose–response increase of cell proliferation and metabolism. In scratch wound and transwell assays, a dose of 20% PL induced a significant increase of wound closure rate at 6 and 24 hrs, and had a strong chemotactic effect. BAPTA‐AM, SB203580 and PD98059 caused 100% inhibition of PL effects, whereas wortmannin reduced to about one third the effect of PL on wound healing and abolished the chemotactic response. Confocal imaging showed the induction by PL of serial Ca2+ oscillations in fibroblasts. Data indicate that cell Ca2+ plays a fundamental role in wound healing even without PL, p38 and ERK1/2 are essential for PL effects but are also activated by wounding per se, PI3K is essential for PL effects and its downstream effector Akt is activated only in the presence of PL. In conclusion, PL stimulates fibroblast wound healing through the activation of cell proliferation and motility with different patterns of involvement of different signalling pathways.

Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein

Background Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. Principal Findings Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca2+-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130–139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1(130–139) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. Conclusion We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25-40% of the cellular calpain activity. In the membrane-bound form the Ca2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles. When both monoclonal antibody and calpain were present in these vesicles, a pronounced inhibition of the membrane bound proteinase activity was observed. These results provide an explanation for the previously observed inhibitory effect of the monoclonal antibody on intracellular calpain activity and on the concomitant inhibition of granule exocytosis. The activated calpain associated with the plasma membrane compartment is therefore identified as the form specifically involved in mediating the physiological responses.

Emerging roles for HMGB1 protein in immunity, inflammation, and cancer.

High-mobility group box 1 (HMGB1) protein is a member of the highly conserved non-histone DNA binding protein family. First identified in 1973, as one of a group of chromatin-associated proteins with high acidic and basic amino acid content, it was so named for its characteristic rapid mobility in polyacrylamide gel electrophoresis. HMGB1 was later discovered to have another function. It is released from a variety of cells into the extracellular milieu to act on specific cell-surface receptors. In this latter role, HMGB1 is a proinflammatory cytokine that may contribute to many inflammatory diseases, including sepsis. Therefore, HMGB1 regulates intracellular cascades influencing immune cell functions, including chemotaxis and immune modulation. The bioactivity of the HMGB1 is determined by specific posttranslational modifications that regulate its role in inflammation and immunity. During tumor development, HMGB1 has been reported to play paradoxical roles in promoting both cell survival and death by regulating multiple signaling pathways. In this review, we focus on the role of HMGB1 in physiological and pathological responses, as well as the mechanisms by which it contributes to immunity, inflammation, and cancer progression.