2D-PAGE reveals the absence of collagenase production in normal

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obtained from bone fractures of otherwise healthy individuals. The osteoblasts were grown for one month in culture using an established method [2] and had ...

59s ZD-PAGE reveals the absence of collagenase production in normal human osteoblasts in culture

DAVID P.HANKEY, W.H.IRWIN McLEAN and ANNE E.HUGHES De artment of Medical Genetics, The Queen’s University of Eelfast, Floor A, City Hospital, Belfast BT9 7AB Osteoblasts are secretory cells which as well as being responsible for bone formation are also central to the initiation and control of bone remodelling. Bone resorption is initiated by collagen breakdown which allows subsequent attachment of the osteoclast (the specialized resorptive cell). Whether or not osteoblasts produce interstitial collagenase is significant in understanding their role in the remodellin sequence. A normafhuman fibroblast cell line F152 [l] was used as a comparison for a number of primary osteoblast cultures obtained from bone fractures of otherwise healthy individuals. The osteoblasts were grown for one month in culture using an established method [2] and had been passaged three times. Osteoblasts cultured for several assa es (over about 5 months) are reported to undergo a Lss of well defined osteoblastic phenotype [3]. Anal sis of secreted roteins in the conditioned media was carriedbut using SDS-{AGE [4] or by 2D-PAGE [l]. [email protected] gels were loaded w t h equivalent counts of [ S]-methionine labelled extracellular rotein. The dried gels were autoradiographed with AmersKam Hyperfilm 0max. The SDS-PAGE results are shown in Fig.lA. The bands representing extracellular colla enase are clearly visible in the fibroblast sample and are atsent from the osteoblast sample. The 2D-PAGE results are shown in Fig.lB (fibroblast secreted protein) and Fig.lC (osteoblast secreted protein). A double train of 1 coproteins (M.wts. 52 and S4kDa) are visible in the fibrotLt sample (boxed in Fig.1B). These have been identified on 2D- els previously by a specific antiserum [5] as the N- and 8-glycoforms of collagenase [6]. All isoforms of the enzyme are found to be absent from the 2D-gels of osteoblast extracellular protein (Fig.lC). In addition, there are a number of other obvious differences between the 2D-gels of fibroblast and osteoblast secreted protein. These proteins are as yet uncharacterised. The results clearly illustrate the absence of secreted collagenase in normal human osteoblasts in culture. From analysis of osteoblasts cultured from a number of individuals this pattern is observed to be consistent. The absence of human collagenase has been demonstrated previously using immuno reci itation, functional assay and Northern blotting bj. &,ever, collagenase secretion has been demonstrated in rodent osteoblast cultures [8,9]. It is proposed that this enzyme, in rodent tissue, is responsible for predis osing the bone surface to subsequent osteoblast attacfment. On the basis of the results obtained here it is concluded that this model may not apply to resorption control in human tissue. It follows that a collagenase may be produced (in human bone at least) by osteoclasts. Absence of collagenase synthesis by human osteoblasts is also of significance in terms of cell lineage, since these cells are thought to be of fibroblastoid descent [ 101. Collagen synthesis and breakdown in connective tissues is a pnmary function of fibroblasts and so it of considerable interest that collagenase, a fibroblast marker, is absent in osteoblasts. Abbreviations: SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electro horesis; 2D-PAGE, twodimensional polyacrylamiL gel electrophoresis

SDS-PAGE. Fibroblast collagenase (arrows).

& (C) 2D-PAGE. Fibroblast collagenase (box).

This work was su ported by a Studentship award from the Department of Egucation for Northern Ireland to D.P.H. We thank Arthritis Research Northern Ireland for additional financial support to D.P.H. We are grateful to Prof.R.A.B.Mollan, De artment of Orthopaedic Surgery, Q.U.B. for help in supp ying the bone samples.


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