Mira Šuša

Coordinated Activation of Notch, Wnt, and Transforming Growth Factor-β Signaling Pathways in Bone Morphogenic Protein 2-induced Osteogenesis Notch TARGET GENE Hey1 INHIBITS MINERALIZATION AND Runx2 TRANSCRIPTIONAL ACTIVITY

To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays. A regulated expression of 394 known genes and 295 expressed sequence tags was detected. The sensitivity and reliability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative reverse transcription-PCR. Functional classification of regulated genes was performed, defining the groups of regulated growth factors, receptors, and transcription factors. The most interesting finding was concomitant activation of transforming growth factor-β, Wnt, and Notch signaling pathways, confirmed by strong up-regulation of their target genes by PCR. The transforming growth factor-β pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed that bone morphogenic protein 2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells, and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved via interaction with Runx2: Hey1 completely abrogated Runx2 transcriptional activity. These findings identify the Notch-Hey1 pathway as a negative regulator of osteoblast differentiation/maturation, which is a completely novel aspect of osteogenesis and could point to possible new targets for bone anabolic agents.

A novel inhibitor of the tyrosine kinase Src suppresses phosphorylation of its major cellular substrates and reduces bone resorption in vitro and in rodent models in vivo

The tyrosine kinase Src has been implicated in the process of osteoclast-mediated bone resorption. Here, we describe a novel class of Src inhibitors, substituted 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines, and characterize one of them, CGP77675, in vitro and in models of bone resorption in vivo. In vitro, CGP77675 inhibited phosphorylation of peptide substrates and autophosphorylation of purified Src (concentration producing half-maximal inhibition [IC50] values 5-20 and 40 nmol/L, respectively). The compound was selective toward other protein kinases: the Src IC50 value was lower than those for Cdc2 (>500-fold), epidermal growth factor (EGF) receptor (7.5-fold), and vascular endothelial growth factor receptor (>50-fold), and for v-Abl (15-fold) and focal adhesion kinase (Fak) (>25-fold). The Src kinase family members Lck and Yes were inhibited with IC50 values 20-fold higher than or equal to Src. To measure the inhibition of cellular Src activity, we identified the major tyrosine-phosphorylated proteins in an Src-overexpressing cell line IC8.1 as Src, Fak, and paxillin. CGP77675 potently inhibited tyrosine phosphorylation of the Src substrates Fak and paxillin, but had much less effect on Src (IC50 values 0.3, 0.5, and 5.7 micromol/L). The phosphorylation of Src in IC8.1 cells reflected phosphorylation of the negative regulatory tyrosine 527 (Y527); thus, the inhibitor was selective against the Y527 C-terminal Src kinase Csk. In osteoblastic MC3T3-E1 cells, CGP77675 inhibited signaling induced by PDGF at the receptor level, but not signaling by EGF, basic fibroblast growth factor, insulin-like growth factor-1, and phorbol 12-myristate 13-acetate. The effect of CGP77675 on bone resorption was evaluated in vitro and in vivo. The parathyroid hormone-induced bone resorption in rat fetal long bone cultures was inhibited with an IC50 of 0.8 micromol/L. CGP77675 dose-dependently reduced the hypercalcemia induced in mice by interleukin-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. Thus, specific Src family kinase inhibitors may be useful for the treatment of diseases associated with elevated bone loss.

Inhibition of Protein Kinase c-Src Reduces the Incidence of Breast Cancer Metastases and Increases Survival in Mice: Implications for Therapy

c-Src is a proto-oncogene, belonging to the nonreceptor protein kinases family, which plays a prominent role in carcinogenesis. In this study, we tested the hypothesis that c-Src could promote breast cancer metastasis acting on several cell types and that pharmacological disruption of its kinase activity could be beneficial for the treatment of metastases. Female BALB/c-nu/nu mice were subjected to intracardiac injection of the human breast cancer cells MDA-MB-231 (MDA-231), which induced prominent bone and visceral metastases. These were pharmacologically reduced by treatment with the c-Src inhibitor [7-{4-[2-(2-methoxy-ethylamino-ethoxy]-phenyl}-5-(3-methoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine] CGP76030 (100 mg/kg/day p.o.), resulting in decreased morbidity and lethality. Metastases were more severe in mice injected with MDA-231 cells stably transfected with wild-type c-Src (MDA-231-SrcWT), whereas transfection in injected cells of a c-Src kinase-dead dominant-negative construct (MDA-231-SrcDN) resulted in reduced morbidity, lethality, and incidence of metastases similar to the mice treated with the inhibitor. An analogous beneficial effect of c-Src inhibition was observed in subcutaneous and intratibial implanted tumors. In vitro, c-Src suppression reduced MDA-231 cell aggressiveness. It also impaired osteoclast bone resorption both directly and by reducing expression by osteoblasts of the osteoclastogenic cytokines interleukin-1β and interleukin-6, whereas parathyroid hormone-related peptide was not implicated. c-Src was also modestly but consistently involved in the enhancement of endothelial cell proliferation in vitro and angiogenesis in vivo. In conclusion, we propose that c-Src disruption affects the metastatic process and thus is a therapeutic target for the treatment of breast cancer.

Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in α-MEM medium, supplemented with 10% of selected fetal calf serum, 1 μM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFκB ligand (RANKL, 50 ng/ml), and transforming growth factor-β1 (TGF-β1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase) and activity (resorbed area and collagen fragments released from bone slices) in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay.

EGF induces biphasic S6 kinase activation: Late phase is protein kinase C-dependent and contributes to mitogenicity

Detailed kinetics reveal that EGF-induced S6 kinase activation is biphasic: an early phase appears at 10-15 min, followed by a late phase between 30 and 60 min. Both activities exhibit the same chromatographic behavior and sensitivity to phosphatase 2A. Direct activation of protein kinase C by TPA induces only late phase activity. Down-regulation of protein kinase C leads to loss of both TPA- and EGF-induced late phase activity, while the early phase is unaffected. The loss of late phase kinase activity results in decreased EGF-induced S6 phosphorylation, protein synthesis, and cell growth. The results indicate that EGF differentially regulates S6 kinase activation by two distinct signaling pathways and that loss of the late or protein kinase C-dependent phase leads to a diminished mitogenic response.

7-Alkyl- and 7-cycloalkyl-5-aryl-pyrrolo[2,3-d]pyrimidines--potent inhibitors of the tyrosine kinase c-Src.

7-Substituted-5-aryl-pyrrolo[2,3-d]pyrimidines have been prepared starting from alpha-bromoacetophenones. These compounds represent a novel class of potent inhibitors of the tyrosine kinase pp60(c-Src) with good specificity towards other tyrosine kinases (EGF-R, v-Abl).

Pyrrolopyrimidine c-Src inhibitors reduce growth, adhesion, motility and invasion of prostate cancer cells in vitro

Two bona fide c-Src inhibitors, denominated CGP77675 and CGP76030, reduced in a time- and concentration-dependent manner (i) the proliferation of the PC3 prostate carcinoma cell line, as assessed by the [3H]-thymidine incorporation test, (ii) the capacity of PC3 cells to adhere and spread on Matrigel substrate, as determined by crystal violet staining, (iii) the ability of PC3 cells to migrate through a gelatine boundary and invade a Matrigel substrate. The latter effect was not due to a decrease of urokinase-type plasminogen activator (uPA), nor of metalloproteinase-2 (MMP-2) activities. The MMP-9 activity, along with the expression of the Tissue Inhibitor of Metalloproteinases (TIMP)-1 and TIMP-2, were reduced by the two inhibitors, consistent with the ability of c-Src to enhance MMP-9 and TIMP expression levels. Collectively, these data demonstrate that the pyrrolopyrimidine-derived c-Src inhibitors significantly reduced PC3 cell activities associated with their malignant phenotype.

Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone

The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. As the role of Hey1 in skeletal physiology is unknown, we analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. Taken together, our data suggest that ubiquitous in vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass.

7-Pyrrolidinyl- and 7-piperidinyl-5-aryl-pyrrolo[2,3-d]pyrimidines--potent inhibitors of the tyrosine kinase c-Src.

7-Heterocyclyl-5-aryl-pyrrolo[2,3-d]pyrimidines represent a new class of highly potent and selective inhibitors of the tyrosine kinase pp60(c-Src).

Substituted 5,7-diphenyl-pyrrolo[2,3d]pyrimidines: potent inhibitors of the tyrosine kinase c-Src.

5,7-Diphenyl-pyrrolo[2,3d]pyrimidines represent a new class of highly potent inhibitors of the tyrosine kinase c-Src (IC50 < 50 nM) with specificity against a panel of different tyrosine kinases. The substitution pattern on the two phenyl rings determines potency and specificity and provides a means to modulate cellular activity.