Expression of a human cartilage procollagen gene (COL2A1) in mouse 3T3 cells.

Expression in a recombinant system has been difficult to obtain for any of the major fibrillar collagens that require processing by eight or more post-translational enzymes. Here, two DNA constructs were designed so that the promoter region of the gene for the pro-alpha 1(I) chain of human type I procollagen drove expression of the human type II procollagen gene in mouse NIH 3T3 cells, a culture line that normally synthesizes type I procollagen but not any cartilage-specific protein such as type II procollagen. Both constructs were expressed as both mRNA and protein. In clones expressing the construct at high levels, the steady-state levels of mRNA and the production of type II procollagen were comparable to the mRNA levels and production of type I procollagen from the endogenous mouse genes. Comparison of clones containing the two constructs demonstrated that sequences extending 80 base pairs beyond the major polyadenylation signal of the gene are not in themselves sufficient for correct termination and 3\u27 processing of RNA transcripts. The results strongly suggest that specific sequences present in a downstream 3.5-kilobase SphI/SphI fragment determine the termination of the transcription. Of special importance is that the system will make it possible to examine the consequences of mutations in the human type II procollagen gene on the processing of RNA transcripts and on the functional properties of the protein simply by using the genomic DNA from leukocytes or other non-cartilaginous sources

A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies.

Canonical Wnt signaling is an osteo-inductive signal that promotes bone repair through acceleration of osteogenic differentiation by progenitors. Dkk-1 is a secreted inhibitor of canonical Wnt signaling and thus inhibits osteogenesis. To examine a potential osteo-inhibitory role of Dkk-1 in osteosarcoma (OS), we measured serum Dkk-1 in pediatric patients with OS (median age, 13.4 years) and found it to be significantly elevated. We also found that Dkk-1 was maximally expressed by the OS cells at the tumor periphery and _in vitro_ Dkk-1 and RANKL are co-expressed by rapidly proliferating OS cells. Both Dkk-1 and conditioned media from OS cells reduces osteogenesis by human mesenchymal cells and by immuno-depletion of Dkk-1, or by adding a GSK3[beta] inhibitor, the effects of Dkk-1 were attenuated. In mice, we found that the expression of Dkk-1 from implanted tumors was similar to the human tumor biopsies in that human Dkk-1 was present in the serum of recipient animals. These data demonstrate that systemic levels of Dkk-1 are elevated in osteosarcoma. Furthermore, the expression of Dkk-1 by the OS cells at the periphery of the tumor probably contributes to its expansion by inhibiting repair of the surrounding bone. These data demonstrate that Dkk-1 may serve as a prognostic or diagnostic marker for evaluation of OS and furthermore, immuno-depletion of Dkk-1 or administration of GSK3[beta] inhibitors could represent an adjunct therapy for this disease

Stanniocalcin-1 Regulates Extracellular ATP-Induced Calcium Waves in Human Epithelial Cancer Cells by Stimulating ATP Release from Bystander Cells

Background: The epithelial cell response to stress involves the transmission of signals between contiguous cells that can be visualized as a calcium wave. In some cell types, this wave is dependent on the release of extracellular trinucleotides from injured cells. In particular, extracellular ATP has been reported to be critical for the epithelial cell response to stress and has recently been shown to be upregulated in tumors in vivo. Methodology/Principal Findings: Here, we identify stanniocalcin-1 (STC1), a secreted pleiotrophic protein, as a critical mediator of calcium wave propagation in monolayers of pulmonary (A549) and prostate (PC3) epithelial cells. Addition of STC1 enhanced and blocking STC1 decreased the distance traveled by an extracellular ATP-dependent calcium wave. The same effects were observed when calcium was stimulated by the addition of exogenous ATP. We uncover a positive feedback loop in which STC1 promotes the release of ATP from cells in vitro and in vivo. Conclusions/Significance: The results indicated that STC1 plays an important role in the early response to mechanical injury by epithelial cells by modulating signaling of extracellular ATP. This is the first report to describe STC1 as a modulator o

Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couette flow cell

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 μL), low-wavelength (down to 180 nm), low-pathlength (100 μm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-β-sheet amyloid fibers of the Alzheimer's derived protein Aβ and the long-chain assemblies of α1-antitrypsin polymers

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/β-catenin signaling

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/β-catenin signaling pathway as indicated by the increased stability and nuclear localization of β-catenin in TIMP-1–deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced β-catenin transcriptional activity, determined by Wnt/β-catenin target gene expression analysis and a luciferase-based β-catenin– activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on β-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1–mediated effects on Wnt/β-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of β-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1–depleted hMSCs and demonstrably reduced axin 2, an antagonist of β-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/β-catenin activity