Differential induction and regulation of matrix metalloproteinases in osteoarthritic tissue and fluid synovial fibroblasts.

OBJECTIVES: To investigate the secretion profiles of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in synovial fluid-derived fibroblasts and to compare them with those of tissue-derived fibroblasts. METHODS: Fibroblast cultures established from synovial tissues (TSC) and fluids (FSC) of the same OA patients were stimulated with tumor necrosis factor(TNF)-alpha, interleukin(IL)-1alpha, IL-1beta, IL-6 and a combination of TNFalpha and IL-1beta. Cocultures of fibroblasts and cartilage were stimulated either with the cytokine combination or with osteoarthritic synovial fluid. Secretion of MMP-1, MMP-3, MMP-8, MMP-13, TIMP-1, and TIMP-2 was measured by enzyme-linked immunosorbent assay. Gelatin zymography and immunoblotting were performed to demonstrate enzyme activity. RESULTS: TNFalpha, IL-1alpha, and IL-1beta led to marked increases in MMP-1 and MMP-3 release (up to 4.2-fold and 547-fold, respectively) by synovial fibroblasts, whereas secretion of MMP-13 was induced by concomitant administration of TNFalpha and IL-1beta. Expression of intracellular MMP-8 was stimulated by cytokines, but adhesion of synovial fibroblasts to cartilage was required for the release. Throughout the study, significantly higher levels of secreted MMPs were observed in stimulated FSC compared to TSC cultures. Furthermore, increases in MMP secretion were not accompanied by increases in secreted TIMP-1 and TIMP-2, resulting in marked imbalances between enzyme and inhibitor levels. CONCLUSIONS: The results provide strong evidence for a significant impact of synovial-derived MMPs on cartilage destruction in OA. In this context, fibroblasts present in the synovial fluid appeared to play an outstanding role.

Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma.

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P <.001). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P <.05) and to a variable degree (up to 5.3-fold; P <.005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6. (Blood. 2000;95:2630-2636)

Ubiquitous expression of the androgen receptor and testis-specific expression of the FSH receptor in the cynomolgus monkey (Macaca fascicularis) revealed by a ribonuclease protection assay.

Androgens are known to exert a variety of effects on an organism while follicle-stimulating hormone (FSH) seems to act specifically on the gonads. To investigate whether these effects are reflected by the expression pattern of the androgen receptor (AR) or the FSH receptor (FSHR) we screened 38 different tissues and organs of one intact and one castrated male non-human primate (Macaca fascicularis). By means of a highly sensitive ribonuclease protection assay (RPA) we demonstrated AR mRNA expression in all tissues of the intact monkey investigated. Immunohistochemistry of selected organs from this monkey revealed a good correlation between AR mRNA and protein expression. In the castrated monkey, the overall AR mRNA expression was markedly lower compared with the intact monkey, although higher expression was present in the pituitary, thyroid and prostate glands. FSHR mRNA was only detected in testicular tissue. This study has revealed, for the first time, ubiquitious expression of the AR mRNA in a non-human primate. The testis-specific expression of the FSHR highlights the importance of FSH for spermatogenesis with the testis being apparently the only target organ.

Quantification of androgen receptor and follicle-stimulating hormone receptor mRNA levels in human and monkey testes by a ribonuclease-protection assay.

A sensitive, solution-hybridization ribonuclease-protection assay (RPA) was established to quantify the expression of mRNA for the androgen receptor (AR) and follicle-stimulating hormone receptor (FSHR) in total RNA samples isolated from tissues of the cynomolgous monkey, human testes obtained from elderly patients undergoing orchidectomy because of prostatic carcinoma or from transsexual men undergoing gender reassignment as well as human cell lines DU 145, REP and RVP. Sensitivity experiments revealed that, in the human and monkey, 1-2 micrograms of total RNA were sufficient to achieve quantifiable signals of the different receptor mRNA species. Quantification of AR and FSHR mRNA levels showed a 1.7-fold higher expression of AR mRNA and a 2.4-fold higher expression of FSHR mRNA in the monkey testes compared to human testes from patients with prostatic carcinoma. Normal spermatogenesis in both human and monkey testes indicated no relationship between spermatogenic status and receptor expression. The significantly lower expression of AR and FSHR mRNA in humans than in monkeys might therefore be either age- or species-related. Quantification of mRNA for AR and FSHR in the testis of the transsexual patients undergoing oestrogen and antiandrogen treatment displayed a drastic increase (4.5-fold) in mRNA for the AR, whereas mRNA for the FSHR was barely detectable. Due to its high sensitivity, reproducibility and its ability to quantify mRNA transcripts, the RPA is a useful tool for investigating expression of low abundant receptor genes and their regulation when only very small amounts of tissue are available. Furthermore, it is suitable for use in clinical and experimental studies in which accurate quantification of transcripts is necessary.

Characterization of the 5' flanking region of the human follicle-stimulating hormone receptor gene.

A genomic clone containing 2.3 kilobases (kb) of the 5' flanking region of the human follicle-stimulating hormone receptor (FSHR) plus the translated region of exon 1 and subsequent sequences of intron A has been isolated and characterized. This portion of the 5' flanking region has neither a TATA nor a CCAAT box and shows features of promoters seen in "housekeeping" genes. Using RNAse protection multiple transcriptional start sites could be identified, the major ones clustered between -114 and -79 bp. Chimeras containing 1486 bp of the 5' flanking region, or deletions thereof, expressed significant chloramphenicol acetyltransferase (CAT) activity when transiently transfected into Chinese hamster ovary (CHO), primary rat Sertoli and human granulosa-lutein cells. Deletion analyses indicated that a proximal promoter can be allocated to the region from -225 to -1 bp.

Molecular cloning of the testicular follicle stimulating hormone receptor of the non human primate Macaca fascicularis and identification of multiple transcripts in the testis.

Reverse transcription PCR was used to amplify the complete open reading frame of the follicle-stimulating hormone receptor (FSHR) from testicular poly (A)+ RNA of the non-human primate Macaca fascicularis. Along with the structural motifs of a G-protein coupled receptor, sequence analysis reveals that the monkey FSHR is highly homologous to the human FSHR and has specific features such as N-linked glycosylation sites which are identical to the human FSHR but not present in the rat or ovine FSHR. Northern blot hybridization of testicular poly (A)+ RNA to a cRNA probe corresponding to the extracellular domain of the monkey FSHR resulted in the identification of several transcripts, indicating alternative splicing events of the primary transcript.