Serglycin Constitutively Secreted by Myeloma Plasma Cells Is a Potent Inhibitor of Bone Mineralization in Vitro

Achilleas D Theocharis,Carina Seidel,Magne Borset,Katalin Dobra,Vadim Baykov,Vassiliki Labropoulou,Ioannis Kanakis,Evangelos Dalas,Nikos K Karamanos,Anders Sundan,Anders Hjerpe

doi:10.1074/jbc.m601061200

Abstract

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n=2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

Highlights

The function of serglycin has not been well studied, but it is likely involved in the packaging of proteins into secretory granules and/or directing the secretion of these molecules [15]
The cDNA for serglycin predicts a molecular size of 14.4 kDa for the mature protein, it should be noted that the core protein of serglycin isolated from various sources exhibits a molecular size of ϳ28 kDa on SDSPAGE even in the presence of ␤-mercaptoethanol
We have shown for the first time that serglycin is the major proteoglycan synthesized and secreted by MM cell lines

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Cultures—The human myeloma cell line JJN3 was a gift from Jennifer Ball (Department of Immunology, University of Birmingham, UK). Serglycin preparations isolated from all four MM cell lines and nonfractionated condition medium as well as bone marrow samples (1 ␮l of bone marrow plasma) from patients with MM and healthy volunteers were diluted in Laemmli sample buffer (Bio-Rad), electrophoresed on a 4% stacking-10% resolving gel, and transferred to Immobilon P membranes at constant current 80 mA at 4 °C for 12 h in 50 mM Tris-HCl, pH 8.3. Immunohistochemistry—Reactivity to the rabbit anti-serglycin antibody and CD138 (syndecan-1) was studied using parallel sections of routinely fixed (4% buffered formalin) and paraffin-embedded bone marrow For this purpose five tissues with MM and five tissues with benign proliferation of plasma cells (including one case with monoclonal gammopathy of unknown significance (MGUS)) were retrieved from the archives at Karolinska University Hospital, Huddinge. PGs were resistant to the action of a mixbecause adsorption phenomena do not interfere with kinetic ture of heparin lyases (figure not shown), but their GAG chains measurements [40]

RESULTS

Cia Serglycin

DISCUSSION

Sundan and Anders Hjerpe