Matrix metalloproteinases-1 and -2, and tissue inhibitor of metalloproteinase-2 production is abnormal in bone marrow stromal cells of multiple myeloma patients

Abstract

We have investigated the production of metalloproteinases (MMP-1, MMP-2, MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in bone marrow stromal cells (BMSCs) of patients with multiple myeloma (MM) and a healthy control. The new findings of this paper is that BMSCs of the MM patients exhibited intrinsic MMP-1, MMP-2 and TIMP-2 overproduction. Production of MMP-1, TIMP-2 and activation of MMP-2 was additionally enhanced in co-cultures of BMSCs with RPMI8226 cells. The ratio between MMP-2 and TIMP-2 was significantly higher in BMSCs of the MM patients than in control. BMSCs of both the control and the MM patients exhibited the presence of MMP-9 latent form, but in co-cultures RPMI8226 cells were the main producers of this metalloproteinase.

Introduction

Matrix metalloproteinases (MMPs) are a family of structurally and functionally related proteinases characterized by the ability to degrade the extracellular matrix (ECM). At least 20 enzymes sharing considerable homology within their major domains were included in the MMP family [1]. Based on their substrate specificity and domain structure, MMPs were divided into four main subgroups. One of these subgroups is represented by gelatinases, also known as type IV collagenases, which degrade gelatine and several types of collagen, with gelatinase A (MMP-2) and gelatinase B (MMP-9) as members. Both of them are involved in tumor initiation, growth, and metastasis [2]. Another class are collagenases and among them the interstitial collagenase (MMP-1), known to initiate degradation of type I collagen in neutral pH. Collagen I is the major structural protein of bone with its degradation being critical for the initiation of bone resorption. After fragmentation by MMP-1, the denatured collagen becomes a substrate for the gelatinases MMP-2 and MMP-9. Thus collagenases and gelatinases work in concert to promote bone resorption [3]. Moreover, MMP-2 and MMP-9 degrade collagen IV, the major constituent of the basement membrane and have been implicated in tumor invasion and metastasis [4].

The activity of MMPs is controlled by their interaction with tissue inhibitors of metalloproteinases (TIMPs). Currently four TIMPs have been identified, most of them being capable to bind and inhibit the activity of all members of the MMP family. Some studies, however, have showed that TIMP-1 binds preferably to MMP-9 and TIMP-2 to MMP-2 [5], [6]. Apart from their role in inhibiting MMPs, TIMPs are known to have growth factor properties, and play a part in stimulation of cell migration and stimulation or inhibition of apoptosis [7], [8]. TIMPs also inhibit angiogenesis via mechanisms that may involve both the inhibition of MMP activity and MMP-independent action [9]. Moreover, TIMP-1 was described as a stromal factor whose high expression is associated with poor clinical outcome in many cancer types [10].

Multiple myeloma (MM) is a B-cell malignancy characterized by the proliferation and accumulation of malignant plasma cells within the bone marrow. Almost all patients with MM have an early excessive bone resorption leading to lytic lesions and to hypercalcemia. Furthermore, although tumor progression is observed mainly within the bone marrow during the early stages of the disease, extramedullary spreading occurs during the terminal stage of the disease. Moreover, malignant cells can be detected in peripheral blood of many patients with MM, suggesting migration of myeloma cells outside the bone marrow [11]. It has already been demonstrated that myeloma cells in co-cultures with bone marrow stromal cells (BMSCs) of healthy subjects can induce up-regulation of MMP-1 and partial conversion of MMP-2 [12] into its active form. Unlike us [13], however, the authors did not detect any MMP-9 production in BMSCs. Moreover, the differences in MMPs production in BMSCs derived from patients with MM and from a healthy control were not examined.

These observations prompted us to address the question of the potential role of MMPs in the changes induced by MM cells in the bone marrow environment of MM patients. We studied MMP-1, MMP-2 and MMP-9 production both by RPMI8226 myeloma cells and bone marrow stromal cells (BMSCs), the latter derived either from MM patients or from healthy controls, cultivated in monocultures or co-cultivated in cell-to-cell contact and non-contact conditions with RPMI8226 cells. We also examined MMP regulation by TIMPs when myeloma cells and BMSCs were cultivated in monocultures or were co-cultured.