title: Hemolin triggers cell survival on fibroblasts in response to serum deprivation by inhibition of apoptosis.
abstract: Fibroblasts are the main cellular component of connective tissues and play important roles in health and disease through the production of collagen, fibronectin and growth factors. Under certain conditions, such as wound healing, fibroblasts intensify their metabolic demand, while the restriction of nutrients affect matrix composition, cell metabolism and behavior. In lepidopterans, wound healing is regulated by ecdysteroid hormones, which upregulate multifunctional proteins such as hemolin. However, the role of hemolin in cell proliferation and wound healing is not clear. rLosac is a recombinant hemolin from the caterpillar Lonomia obliqua whose proliferative and cytoprotective effects on endothelial cells have been described. Here, we show that rLosac induces a marked cell survival effect on fibroblast submitted to serum deprivation, which is observable as early as 24h, as demonstrated through the MTT assay, as well as an increase in migration of human dermal fibroblasts (HDF). No effects on cell proliferation or cell cycle distribution of fibroblasts in normal conditions were observed, suggesting that rLosac induces an effect in stressful conditions such serum deprivation but not when nutrient are sufficient. By flow cytometry, rLosac caused an apparent dose-dependent increase in cells in the S phase of the cell cycle and a significant reduction of cells with fragmented DNA. Furthermore, treatment with rLosac results in a significant decrease in the production of reactive oxygen species and in the loss of mitochondrial membrane potential, indicating that a reduction in oxidative stress is involved in rLosac-mediated cytoprotection. Our results also show an up-regulation of Bcl-2 and a down-regulation of Bax protein levels, inhibition of cytochrome c release and a reduction in caspase-3 levels, all considered critical factors for apoptosis. Moreover, rLosac treatment reduces the morphological changes induced by prolonged serum deprivation including the emergence of apoptotic bodies, nucleus fragmentation, cytoplasmic vacuolization and loss of extracellular matrix organization. The wound scratch test assay revealed that rLosac could enhance wound healing in vitro. Altogether, these findings suggest that rLosac strongly induces cellular protection in conditions of stress by serum deprivation preventing damage and loss of mitochondrial function by inhibiting apoptosis. This finding opens a new perspective to further understand the role of hemolin proteins during cellular processes such as wound healing and development.
authors.affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Rosemary Viola
authors.initials: RV
authors.lastname: Bosch
authors.affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP, Brazil; Center of Research in Toxins, Immune Response and Cell Signaling (CETICS), Butantan Institute, São Paulo, SP, Brazil; Center of Excellence in the New Target Discovery, Butantan Institute, São Paulo, SP, Brazil. Electronic address: miryam.flores@butantan.gov.br.
authors.forename: Miryam Paola
authors.initials: MP
authors.lastname: Alvarez-Flores
authors.affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Durvanei Augusto
authors.initials: DA
authors.lastname: Maria
authors.affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP, Brazil; Center of Research in Toxins, Immune Response and Cell Signaling (CETICS), Butantan Institute, São Paulo, SP, Brazil; Center of Excellence in the New Target Discovery, Butantan Institute, São Paulo, SP, Brazil. Electronic address: ana.chudzinski@butantan.gov.br.
authors.forename: Ana Marisa
authors.initials: AM
authors.lastname: Chudzinski-Tavassi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D020405:Dermis / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D007136:Immunoglobulins / Q000494:pharmacology*; D019476:Insect Proteins / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010447:Peptide Hydrolases / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; D007136:Immunoglobulins; D019476:Insect Proteins; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C066846:hemolin; C557790:Losac protein, Lonomia obliqua; D010447:Peptide Hydrolases; D053148:Caspase 3
keywords: Apoptosis; Cell survival; Fibroblast; Hemolin; Serum deprivation; Wound healing