Editorial Type:
Molecular Biology

Distribution and processing of a disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae

Erica Pawlak Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003.

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 BS
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Le Wang Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003.

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 PhD
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Philip J. Johnson Department of Equine Internal Medicine, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Gerard Nuovo Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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Almaz Taye Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003.

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James K. Belknap Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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Dominique Alfandari Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003.

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Samuel J. Black Department of Veterinary and Animal Sciences, College of Natural Sciences, University of Massachusetts, Amherst, MA 01003.

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DOI:
https://doi.org/10.2460/ajvr.73.7.1035
Volume/Issue:
Volume 73: Issue 7
Online Publication Date:
01 Jul 2012
Restricted access
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Abstract

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.

Sample—Laminae from the forelimb hooves of 8 healthy horses.

Procedures—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 and Clinical Relevance—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.

Abstract

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.

Sample—Laminae from the forelimb hooves of 8 healthy horses.

Procedures—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 and Clinical Relevance—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.

Contributor Notes

Supported by the Morris Animal Foundation (D08EQ-054) and the USDA National Research Initiative Cooperative State Research, Education, and Extension Service (2007-35204-18313).

Dr. Black was supported by the USDA National Research Initiative Cooperative State Research, Education, and Extension Service (MAS00907).

Dr. Alfandari was supported by the National Institutes of Health (DE016289).

Ms. Pawlak was supported by a Lotta M. Crabtree Fellowship in Agriculture.

Ms. Taye was supported by a fellowship from the Northeast Alliance for Graduate Education and the Professoriate.

Presented, in part, at the American Association of Equine Practitioners Foundation Equine Laminitis Workshop, West Palm Beach, Fla, November 2009, and at the Conference for Research Workers in Animal Diseases, Chicago, November 2010.

The authors thank Dr. Andria Cogswell for assistance with acquisition of laminae samples, Aileen Thomas for assistance with vaccination of the rabbits, and Baixang Zou for technical assistance.

Ms. Pawlak and Ms. Wang contributed equally to the study.

Address correspondence to Dr. Black (sblack@vasci.umass.edu).
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Publication Date:
01 Jul 2012

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