Modeling the cholesteatoma microenvironment: coculture of HaCaT keratinocytes with WS1 fibroblasts induces MMP-2 activation, invasive phenotype, and proteolysis of the extracellular matrix.
BACKGROUND
Increased keratinocyte proliferation, increased keratinocyte migration, elaboration of proteases resulting in proteolysis of the extracellular matrix (ECM), and destruction of surrounding tissues all typify the course of cholesteatoma growth. The contribution of stromal fibroblasts to these behaviors remains relatively unexplored.


OBJECTIVE
Our objective for the current studies was to create a simple model with which to study these cholesteatoma behaviors, specifically, cell migration, invasion, and proteolysis of the extracellular matrix as well as the role of fibroblasts in the activated keratinocyte phenotype of cholesteatoma.


METHODS
The authors conducted an in vitro culture model.


METHODS
The resulting model consists of activated keratinocytes (HaCaT cells) cocultured with normal dermal fibroblasts (WS1 cells) within a three-dimensional reconstituted ECM. We used a confocal imaging assay and software analysis to quantify total functional proteolysis of the ECM in monotypic and organotypic cocultures. This was accomplished by growing cells on an artificial ECM comprised of Matrigel and DQ-collagen IV. DQ-collagen is a "quenched" fluorescent peptide whose fluorescence is unmasked by proteolytic cleavage.


RESULTS
Organotypic cocultures of keratinocytes and fibroblasts exhibited increased cell migration, increased cell invasion, increased matrix metalloproteinase-2 secretion and activation, and increased proteolysis of type IV collagen in three-dimensional ECM. Exposure to NSC27366, inhibitor of the small GTPase, Rac, resulted in reduction in both cell invasion and ECM proteolysis.


CONCLUSIONS
Stromal fibroblasts may stimulate the invasive phenotype of keratinocytes, including ECM proteolysis. Increased cell invasion and proteolysis are dependent on the Rac pathway in this model. This simple culture model may help further our understanding of these destructive behaviors in cholesteatoma keratinocytes.
Laeeq
Sabahat
S
Department of Otolaryngology, Children's Hospital of Michigan, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Faust
Russell
R
D002450:Cell Communication; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D002781:Cholesteatoma / Q000473:pathology / Q000503:physiopathology*; D018920:Coculture Techniques; D003094:Collagen / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D004338:Drug Combinations; D004789:Enzyme Activation / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D007797:Laminin / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D018613:Microscopy, Confocal; D010641:Phenotype; D011509:Proteoglycans / Q000378:metabolism; D017154:Stromal Cells / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors
D024141:Collagen Type IV; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020830:rac1 GTP-Binding Protein