Distribution and processing of a disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae.
OBJECTIVE
To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses.


METHODS
Laminae from the forelimb hooves of 8 healthy horses.


METHODS
Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization.


RESULTS
Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae.


CONCLUSIONS
Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.
Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Erica
E
Pawlak
Le
L
Wang
Philip J
PJ
Johnson
Gerard
G
Nuovo
Almaz
A
Taye
James K
JK
Belknap
Dominique
D
Alfandari
Samuel J
SJ
Black
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D020869:Gene Expression Profiling / Q000662:veterinary; D006724:Hoof and Claw / Q000378:metabolism*; D006736:Horses / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry / Q000662:veterinary; D011348:Procollagen N-Endopeptidase / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D053675:Versicans / Q000235:genetics / Q000378:metabolism*
D053674:Aggrecans; D053675:Versicans; D012313:RNA; D006820:Hyaluronic Acid; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein