Ikk2-mediated inflammatory activation of arterial endothelial cells promotes the development and progression of atherosclerosis.

BACKGROUND AND AIMS: Inflammatory activation of endothelial cells is considered to be the first step in the development of atherosclerosis. Here, we determined the consequences of chronic endothelial activation via the NF-κB activator Ikk2 (Inhibitor of nuclear factor kappa-B kinase 2, Ikk-beta) on the development and progression of atherosclerosis. METHODS: We established a conditional transgenic mouse model, expressing a tamoxifen-inducible, constitutively active form of Ikk2 exclusively in arterial endothelial cells (caIkk2EC mice) on an ApoE-deficient background. Mice were fed a Western-type diet and endothelial Ikk2 was activated either at early or late stages of atherosclerosis. RESULTS: En face preparations of isolated aortas revealed a significant increase in plaque area in caIkk2EC mice at 12 weeks of Western-type diet as compared to ApoE-deficient littermates. This was accompanied by increased infiltration of macrophages and T cells into the lesion. Several chemokine/cytokine and immune cell pathways were significantly upregulated in the aortic transcriptome of caIkk2EC mice. Of note, in mice with established atherosclerosis, activation of endothelial Ikk2 still further accelerated progression of atherosclerosis. This indicates that inflammatory endothelial activation is crucial during all stages of the disease. CONCLUSIONS: Our results show for the first time that chronic inflammatory activation of arterial endothelial cells accelerates the development and progression of atherosclerosis both at early and late stages of disease development. Thus, pharmacological targeting of endothelial inflammation emerges as a promising treatment approach.

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin.

The increased Sprouty4 expression in response to serum is transcriptionally controlled by Specific protein 1.

Sprouty proteins control length and intensity of the intracellular signal transduction cascade activated by mitogens in the cellular environment. As part of a negative feedback loop, their expression is supposed to be elevated by the same factors. In this report, Sprouty4 expression in response to serum and the underlying regulatory mechanisms were investigated. We verified that Sprouty4 expression is activated by serum addition in all tested cells independent of their origin. Strict correlation between Sprouty4 protein levels and promoter activity indicates mainly transcriptional regulation of Sprouty4 serum-responsiveness. Induction of the mitogen-activated protein kinase pathway is required for Sprouty4 promoter activation in the presence of serum. Nonetheless, signal transduction via this pathway is not sufficient to fully induce the Sprouty4 promoter. Instead, deletion and mutation analysis identified two annotated Specific protein 1 binding sites as the critical cis-elements responsible for conferring the serum induction of the promoter. Corroborating, repressed Specific protein 1 activity or levels result in constitutive lowered transcriptional activity of the Sprouty4 promoter. These data demonstrate that Specific protein 1 plays a crucial role in the regulation of Sprouty4 in response to serum.

Expression of microRNA-21 in non-small cell lung cancer tissue increases with disease progression and is likely caused by growth conditional changes during malignant transformation.

MicroRNAs can govern up to hundred different mRNAs and are important regulators of gene expression programs in development and disease. We analyzed the expression of microRNA-21, one of the most common oncomirs, in non-small cell lung cancer (NSCLC). Using northern blots the microRNA-21 expression levels of NSCLC-derived tissue and cell lines were measured. In line with earlier observations we show that mature microRNA-21 expression levels are highly increased in NSCLC-derived tissue compared to normal lung tissue. Additionally, we demonstrate that microRNA-21 levels correlate with malignancy since its expression in higher staged tumors is significantly more elevated compared to stage 1A. Interestingly, microRNA-21 levels in cultured NSCLC-derived cells are comparable to the expression detected in non-malignant lung tissue. Since microRNA-21 levels showed no fluctuation during the cell cycle, accelerated proliferation of tumor cells is not responsible for microRNA-21 upregulation in the tumor compartment. Similarly to NSCLC-derived cancer cells, the tumor-associated fibroblasts show low expression levels of microRNA-21. Together, these data indicate that rather microenviromental and growth conditional changes than intrinsic features of the cancer cells are responsible for the observed increase of microRNA-21 levels in tumor tissues. Subsequently culturing conditions were changed to assess the impact of co-cultivation with fibroblasts, hypoxia and anchorage-independent growth on microRNA-21 expression. While co-cultivation with tumor-associated fibroblasts had no effect on microRNA-21 expression, both hypoxia and anchorage-independent growth cause a microRNA-21 elevation. In summary, our data demonstrate that growth conditions especially expected in more malignant tumors result in microRNA-21 upregulation explaining the observed increase in higher staged lung cancer tissue, but not in lung cancer-derived cells.

Sprouty2 but not Sprouty4 is a potent inhibitor of cell proliferation and migration of osteosarcoma cells.

As negative regulators of receptor tyrosine kinase-mediated signalling, Sprouty proteins fulfil important roles during carcinogenesis. In this report, we demonstrate that Sprouty2 protein expression inhibits cell proliferation and migration in osteosarcoma-derived cells. Although earlier reports describe a tumour-promoting function, these results indicate that Sprouty proteins also have the potential to function as tumour suppressors in sarcoma. In contrast to Sprouty2, Sprouty4 expression failed to interfere with proliferation and migration of the osteosarcoma-derived cells, possibly due to a less pronounced interference with mitogen-activated protein kinase activity. Sequences within the NH2-terminus are responsible for the specific inhibitory function of Sprouty2 protein.

Bimodal expression of Sprouty2 during the cell cycle is mediated by phase-specific Ras/MAPK and c-Cbl activities.

Sprouty2 is an important inhibitor of cell proliferation and signal transduction. In this study, we found a bimodal expression of Sprouty2 protein during cell cycle progression after exit from quiescence, whereas elevated Sprouty4 expression in the G1 phase stayed high throughout the rest of the cell cycle. Induction of the mitogen-activated protein kinase via activated Ras was crucial for increased Sprouty2 expression at the G0/G1 transition. Following the first peak, accelerated proteasomal protein degradation caused a transient attenuation of Sprouty2 abundance during late G1. Since the decline in its expression was abolished by dominant negative c-Cbl and the timely restricted interaction between Sprouty2 and c-Cbl disappeared at the second peak of Sprouty2 expression, we conclude that the second phase in the cell cycle-specific expression profile of Sprouty2 is solely dependent on ubiquitination by c-Cbl. Our results suggest that Sprouty2 abundance is the result of strictly coordinated activities of Ras and c-Cbl.

Sprouty4 levels are increased under hypoxic conditions by enhanced mRNA stability and transcription.

Sprouty (Spry) proteins are well-known negative regulators of receptor tyrosine kinase-mediated signalling. Their expression is controlled by mitogens, implying a negative feedback loop. Correspondingly, the different members of the family fulfil important roles during organogenesis by adjustment of growth factor-induced processes. In addition, Spry4, one member of this protein family, has been shown to regulate angiogenesis by inhibiting vascular endothelial cell growth factor-induced extracellular signalling-regulated kinase (ERK) activation. Because oxygen is an important regulator of angiogenesis, we investigated Spry4 expression patterns under hypoxic conditions. Our data demonstrate that both hypoxia and desferrioxamine (DFO) treatment increased Spry4 expression. Following iron depletion, elevated Spry4 levels were detected in several cell types independent of tissue origin, presence of mitogens, cell differentiation and malignancy. Evaluation of the underlying regulative mechanisms revealed that augmented transcription and increased mRNA stability enhance mRNA levels of Spry4 in response to DFO. This study unveils a growth factor-independent regulation mechanism of Spry4 expression. Because increased Spry4 levels are accompanied by disappearing ERK phosphorylation, Spry4 might be involved in the timely restriction of MAPK signals under hypoxic conditions, similar to its role in mitogen-regulated processes. However, the functional significance of the observed upregulation of Spry4 during iron depletion remains to be clarified.