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Our laboratory is interested in delineating normal tissue homeostasis and understanding pathological changes during tissue repair and cancer. The major themes in the laboratory are the biological and molecular mechanisms of dysregulation of pathways involved in cell growth, migration, invasion, and intercellular communication during tumor development and progression. We are then exploring strategies for therapy of tumors and tissue damage.
Keratinocytes in the epidermis of the skin control proliferation of melanocytes and dictate which cell surface molecules are expressed for adhesion and migration. The lab's working hypothesis is that melanocyte proliferation is possible if they decouple from keratinocytes by down-regulating E-cadherin and its co-receptor desmoglein-1, which will interrupt gap junctions. Down-regulation of expression of the cell-cell communication molecules is mediated through the production of HGF, ET-1, and PDGF. The melanocytes then retract their dendrites that had connected them to keratinocytes of the suprabasal layers by activating rac and rho genes. Cell division is likely initiated through activation by keratinocyte-derived growth factorssuch as bFGF, SCF, ET-1 or by fibroblast-derived factors such as HGF, IGF-1, or ET-3. After cell division, melanocytes separate and glide over the basement membrane using integrins such as alpha6ß1 or alpha7ß1 before repositioning singly among basal layer keratinocytes.
Using the three-dimensional organotypic culture model of human skin consisting of dermis and epidermis we are retracing each step in the proliferation cascade of melanocytes to better understand dysregulation of growth and cell-cell communication in melanoma. The lab uses adenoviral and lentiviral vectors to transfer genes for overexpressing or inhibiting a function of interest.
The unique model of human skin reconstruction in vitro and in vivo allows us to investigate signaling between melanocytes and keratinocytes for tissue homeostasis and its dysregulation during transformation to nevi and melanomas. Melanoma cells have escaped from keratinocytes by downregulating E-cadherin and upregulating N-cadherin. The cadherin switch allows a cell partner change because now melanoma cells can adhere through N-cadherin to fibroblasts and endothelial cells. Overexpression of E-cadherin in melanoma cells allows keratinocytes to adhere to them and regain control over proliferation and the expression of cell surface molecules, so the malignant cells revert to a non-malignant phenotype.
The team expects in the next few years to identify and characterize transcriptional activators or repressors in normal melanocytes that are non-functional in melanoma cells but can be reactivated to control growth and invasion.
Fukunaga-Kalabis, M., Martinez, G., Liu, Z-J., Kalabis, J., Mrass, P., Weninger, W., Firth, S.M., Planque, N., Perbal, B., Herlyn, M.: CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1. J Cell Biol. 175: 563-569, 2006. PMID 17101694
Melanocyte homeostasis in normal human skin. Cell division for melanocytes likely begins with the upregulation of growth factors produced by either fibroblasts or keratinocytes, which lead to down-regulation of cadherins and decoupling of melanocytes from keratinocytes. Mitogenesis is driven by other growth factors from dermis or epidermis. Migration for repositioning, anchorage to the basement membrane, re-coupling to keratinocytes, dendrite extension into the upper cell layers, and growth control by keratinocytes complete the cycle for melanocytes to maintain a stable ratio with epidermal keratinocytes. Repositioning of melanocytes to the new location on the epidermal basal layer and their re-coupling with keratinocytes is intitated with cadherin mediated binding.
Human skin reconstructs and their grafting to mice. Human skin can be rebuilt in vitro by first embedding fibroblasts in collagen and then layering on top melanocytes and keratinocytes (34, in press). When the keratinocytes are exposed to air, they differentiate to form multiple layers. Skin reconstructs are being used in medicine for covering wounds of patients. Skin reconstructs show clearly the effects of dysbalance in homeostasis if growth factors are overexpressed prior to inclusion of cells in the reconstructs. bFGF expressed in melanocytes leads to poor adhesion of the melanocytes to the basement membrane and stimulation of the fibroblasts. The dysregulation of the homeostatic balance is more pronounced if SCF is overexpressed in keratinocytes whereas melanocytes were transduced with the bFGF genes.
While skin reconstructs in vitro have a maximum life-span of approximately 1 month, survival is increased to several months or years when grafted to living hosts. Figure shows a pigmented human skin reconstruct on an immunodeficient SCID mouse. Histological and immunohistochemical analyses revealed that i) host vessels and immune cells infiltrated the dermis of the grafts, ii) a papillary morphology of the upper dermis developed, iii) the differentiation of the epidermis improved, and iv) the basement membrane matured. Likely, not yet defined diffusible factors from the host's microenvironment support the morphology and viability of the grafted human skin reconstructs. Fibroblasts that are transduced with bFGF and embedded in collagen produce increased levels not only of bFGF but also of other growth factors that are mitogens for melanocytes, for example ET-3. If ET-3 is overexpressed in fibroblasts, EGF receptor and neuropilin are upregulated as determined by microarray experiments, whereas chloride channel proteins, matrix metalloproteinases, and matrix proteins are downregulated suggesting that the induction of ET-3 leads to a complex cascade of gene activation and inactivation with potential consequences for melancoyte adhesion to the basement membrane. The rationale for switching the transformation model from intact skin to skin reconstructs is provided in the Design where we also discuss potential pitfalls and alternatives.
Melanocytes in skin reconstructs. (A) Melanocytes overexpressing bFGF. Cells detach from the basement membrane. Arrow indicate melanocytes in epidermis. Note also stimulation of fibroblast proliferation. (B) Melanocytes transduced with LacZ as control. (C) Melanocytes transduced with bFGF and keratinocytes transduced with SCF. The overexpression of the two growth factors in two cell types leads to increased melanocyte proliferation and their dissociation from the basement membrane. (D) Melanocytes and keratinocytes transduced with LacZ as control.
Human skin reconstructs grafted to SCID mice. (A) Pigmented human skin reconstruct 1 month after grafting. (B-D) Histological sections of human skin reconstruct 39 days after grafting. (B) H&E staining. (C) Stain for collagen IV in red demonstrates a mature basement membrane at the epidermal-dermal junction and the walls of murine vessels, which have invaded into the human dermis. (D) Stain for S100 in red shows the melanocytes in the basal layer of the human epidermis.
Li, L., Fukunaga-Kalabis, M., Herlyn, M.: The three-dimensional human skin reconstruct model: a tool to study normal skin and melanoma progression. J. Vis. Exp. Aug 3; 54.pli 2937. doi: 10.3791/2937, 2011. http://www.jove.com/details.php?id=2937&status=a4943k. PMID 21847077
The microscope in the image belonged to William E. Horner, M.D., a collaborator with Caspar Wistar, M.D., in the early 1800s.
Dr. Horner, a lecturer at the University of Pennsylvania, was a pioneer of the use of microscopes in anatomical and medical research. He authored Special Anatomy and Histology, a seminal text on the subject.