Bisphosphonates modulate vital functions of human osteoblasts and affect their interactions with breast cancer cells.

Bisphosphonates (BPs) are in clinical use for the treatment of breast cancer patients with bone metastases. Their anti-resorptive effect is mainly explained by inhibition of osteoclast activity, but recent evidence also points to a direct action of BPs on bone-forming osteoblasts. However, the mechanisms how BPs influence osteoblasts and their interactions with breast cancer cells are still poorly characterized. Human osteoblasts isolated from bone specimens were characterized in depth by their expression of osteogenic marker genes. The influence of the nitrogen-containing BPs zoledronate (Zol), ibandronate (Iban), and pamidronate (Pam) on molecular and cellular functions of osteoblasts was assessed focusing on cell proliferation and viability, apoptosis, cytokine secretion, and osteogenic-associated genes. Furthermore, effects of BPs on osteoblast-breast tumor cell interactions were examined in an established in vitro model system. The BPs Zol and Pam inhibited cell viability of osteoblasts. This effect was mediated by an induction of caspase-dependent apoptosis in osteoblasts. By interfering with the mevalonate pathway, Zol also reduces the proliferation of osteoblasts. The expression of phenotypic markers of osteogenic differentiation was altered by Zol and Pam. In addition, both BPs strongly influenced the secretion of the chemokine CCL2 by osteoblasts. Breast cancer cells also responded to Zol and Pam with a reduced cell adhesion to osteoblast-derived extracellular matrix molecules and with a decreased migration in response to osteoblast-secreted factors. BPs revealed prominent effects on human osteoblasts. Zol and Pam as the most potent BPs affected not only the expression of osteogenic markers, osteoblast viability, and proliferation but also important osteoblast-tumor cell interactions. Changing the osteoblast metabolism by BPs modulates migration and adhesion of breast cancer cells as well.

Release of matrix metalloproteinase-8 during physiological trafficking and induced mobilization of human hematopoietic stem cells.

Previous studies indicate that the release of proteases, including the gelatinase matrix metalloproteinase (MMP)-9, from mature granulocytes plays a crucial role in cytokine-induced hematopoietic stem and progenitor cell (HSPC) mobilization. However, studies with MMP-9-deficient mice revealed that HSPC mobilization was normal in these animals, suggesting that additional proteases must be active at clinically relevant cytokine concentrations. In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. During granulocyte colony-stimulating factor-induced HSPC mobilization, highly elevated serum concentrations of MMP-8 were observed on days 4 to 6 of the mobilization regimen, concomitantly with elevated MMP-9 serum levels and higher numbers of circulating CD34(+) cells. Elevated serum concentrations of both proteases were also found in umbilical cord blood serum. In functional assays, adhesion of HSPC to osteoblasts as an essential component of the endosteal stem cell niche is negatively influenced by MMP-8. The chemokine CXCL12, which is critically involved in stem cell trafficking, can be proteolytically processed by MMP-8 treatment. This degradation has a strong inhibitory influence on HSPC migration. Taken together, our data strongly suggest that MMP-8 can be directly involved in hematopoietic stem cell mobilization and trafficking.

Osteoblast-secreted factors enhance the expression of dysadherin and CCL2-dependent migration of renal carcinoma cells.

Renal cell carcinoma (RCC) frequently metastasizes to the bone marrow. These metastases are characterized by extensive osteolytic lesions. The mechanism, however, by which RCC cells metastasize to bone marrow remains poorly understood. To unravel the role of bone marrow cells in this context, we performed cell adhesion and migration assays using human RCC cell lines to analyze the influence of resident bone marrow cells on renal tumor cells. The strongest adhesion of RCC cells was observed to osteoblasts. Moreover, conditioned medium of osteoblasts (OB-CM) significantly increased RCC cell migration. By gene expression analysis dysadherin was identified as a transcript whose expression could be elevated more than twofold in RCC cells when exposed to OB-CM. Suppression of dysadherin expression in RCC cells by siRNA reduced their ability to migrate in the presence of OB-CM. Furthermore, the RCC cells secreted high amounts of the chemokine CCL2 when tumor cells migrated under the influence of osteoblast-secreted factors. CCL2 neutralization strongly reduced the migratory ability of the RCC cells. Silencing the expression of dysadherin in RCC cells resulted in a twofold reduction of CCL2 protein expression indicating a dysadherin-dependent expression of the chemokine. Taken together, our data show that osteoblasts are the major cell type of the bone marrow that affect RCC cells by secreting factors that increase the expression of dysadherin and CCL2 in the tumor cells leading to enhanced cell migration. These data suggest an osteoblast-induced autocrine mechanism for a facilitated homing of RCC cells to the bone marrow.

The integrin alpha9beta1 on hematopoietic stem and progenitor cells: involvement in cell adhesion, proliferation and differentiation.

BACKGROUND: Hematopoietic stem and progenitor cells can interact with their microenvironment via integrins which are adhesion receptors consisting of alpha and beta subunits. Current knowledge suggests that the integrin subunits alpha4 and alpha6 expressed on hematopoietic stem and progenitor cells have distinct roles in retaining stem cells in the bone marrow. The aim of our study was to gain insight into the expression and functions of the integrin subunits alpha7-alpha11 within the endosteal stem cell niche. DESIGN AND METHODS: Human osteoblasts isolated from trabecular bone and hematopoietic stem and progenitor cells purified from umbilical cord blood or bone marrow aspirates were analyzed for the expression of integrin alpha7-alpha11 chains by reverse transcriptase polymerase chain reaction. The involvement of the integrin alpha9beta1 in hematopoietic stem and progenitor cell adhesion, proliferation and differentiation was analyzed in functional assays. RESULTS: Transcripts for all investigated integrin chains were found in primary osteoblasts. Highly purified hematopoietic stem and progenitor cells, however, expressed only transcripts encoding integrin subunits alpha7 and alpha9. Flow cytometric analysis verified extracellular expression of the integrin alpha9beta1 on hematopoietic stem and progenitor cells. Cell-cell adhesion assays with osteoblasts and dye-labeled CD34(+) hematopoietic stem and progenitor cells in the presence of function-blocking antibodies revealed a role of integrin alpha9 in hematopoietic stem and progenitor cell adhesion to osteoblasts. Furthermore, the addition of anti-integrin alpha9 antibodies significantly inhibited proliferation and in vitro differentiation of CD34(+) hematopoietic stem and progenitor cells. CONCLUSIONS: The integrin alpha9beta1 has been identified as a new member of the integrin beta1-subfamily expressed on human hematopoietic stem and progenitor cells. The functional studies strongly suggest that integrin alpha9beta1 contributes to adhesion and differentiation of hematopoietic stem and progenitor cells in the endosteal stem cell niche.