In vitro model for penetration of sensory nerve fibres on a Matrigel basement membrane: implications for possible application to intractable pruritus.
BACKGROUND
Epidermal hyperinnervation occurs in dermatoses with intractable pruritus, such as atopic dermatitis, suggesting that the hyperinnervation is partly responsible for abnormal itch perception.


OBJECTIVE
To investigate the mechanisms of penetration of sensory nerve fibres into the basement membrane of the skin.


METHODS
A rat dorsal root ganglion neurone culture system consisting of Matrigel and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. In some experiments, matrix metalloproteinase (MMP) blockers and semaphorin 3A (Sema3A) were added to the culture system. Matrigel-coated membranes were stained with anti-Tau antibody, and the number of nerve fibres that crossed the membrane was counted. Expression of MMPs in the cultured neurones was examined at mRNA and protein levels by quantitative reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. The activity was also examined by zymography.


RESULTS
Nerve fibres penetrated into Matrigel in the presence of an NGF concentration gradient, which was dose-dependently inhibited by GM6001, a broad-spectrum MMP inhibitor. Transcripts for MMP2, but not MMP9, were increased in the cultured neurones, and the penetration was dose-dependently inhibited by MMP-2 blockers. MMP-2 and its activity were partially localized on the NGF-responsive growth cones. NGF also upregulated pro-MMP-2 activation molecules in the cultured neurones. Sema3A stimulation showed the opposite effects on these NGF-dependent events. Interestingly, MMP2 expression was modulated by extracellular matrix (ECM) substrates for this enzyme.


CONCLUSIONS
Membrane-associated MMP-2 contributes to penetration of nerve fibres into Matrigel through modulation by axonal guidance molecules and/or ECM. These findings provide insight for understanding the development of intractable pruritus involving epidermal nerve density.
Tominaga
M
M
Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan.
Kamo
A
A
Tengara
S
S
Ogawa
H
H
Takamori
K
K
D000818:Animals; D001485:Basement Membrane / Q000294:innervation*; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D004151:Dipeptides / Q000494:pharmacology; D004338:Drug Combinations; D005727:Ganglia, Spinal / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D007797:Laminin; D020781:Matrix Metalloproteinase 1 / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D011509:Proteoglycans; D011537:Pruritus / Q000209:etiology*; D012313:RNA / Q000378:metabolism; D051381:Rats; D040121:Semaphorin-3A / Q000494:pharmacology
D003470:Culture Media; D004151:Dipeptides; D004338:Drug Combinations; D007797:Laminin; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011509:Proteoglycans; D040121:Semaphorin-3A; C059485:matrigel; D012313:RNA; D003094:Collagen; D020932:Nerve Growth Factor; D020782:Matrix Metalloproteinases; D020781:Matrix Metalloproteinase 1