Effects of cleavage by a disintegrin and metalloproteinase with thrombospondin motifs-4 on gene expression and protein content of versican and aggrecan in the digital laminae of horses with starch gruel-induced laminitis.
OBJECTIVE
To determine whether increased gene expression of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) in laminae of horses with starch gruel-induced laminitis was accompanied by increased enzyme activity and substrate degradation.


METHODS
Laminae from the forelimb hooves of 8 healthy horses and 17 horses with starch gruel-induced laminitis (6 at onset of fever, 6 at onset of Obel grade 1 lameness, and 5 at onset of Obel grade 3 lameness).


METHODS
Gene expression was determined by use of cDNA and real-time quantitative PCR assay. Protein expression and processing were determined via SDS-PAGE and quantitative western blotting. Protein distribution and abundance were determined via quantitative immunofluorescent staining.


RESULTS
ADAMTS-4 gene expression was increased and that of versican decreased in laminitic laminae, compared with expression in healthy laminae. Catalytically active ADAMTS-4 also was increased in the tissue, as were ADAMTS-4-cleavage fragments of versican. Immunofluorescent analyses indicated that versican was depleted from the basal epithelia of laminae of horses at onset of Obel grade 3 lameness, compared with results for healthy laminae, and this was accompanied by regional separation of basal epithelial cells from the basement membrane. Aggrecan gene and protein expression were not significantly affected.


CONCLUSIONS
Changes in gene and protein expression of ADAMTS-4 and versican in the basal epithelium of laminitic laminae indicated a fundamental change in the physiology of basal epithelial cells. This was accompanied by and may have caused detachment of these cells from the basement membrane.
Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Le
L
Wang
Erica
E
Pawlak
Philip J
PJ
Johnson
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; D004789:Enzyme Activation; D005534:Foot Diseases / Q000235:genetics / Q000378:metabolism / Q000662:veterinary*; D020869:Gene Expression Profiling / Q000662:veterinary; D006724:Hoof and Claw / Q000378:metabolism*; D006734:Horse Diseases / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D011348:Procollagen N-Endopeptidase / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D018709:Statistics, Nonparametric; D053675:Versicans / Q000235:genetics / Q000378:metabolism*
D053674:Aggrecans; D053675:Versicans; D012313:RNA; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein