RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene-and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediatordependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.activator ͉ transcription ͉ repression ͉ negative regulator

We have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation.

Objective LRP1 is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells (SMC). Mice in which the lrp1 gene is deleted in SMC (smLRP1-/-) on an LDLr-deficient background display excessive PDGF signaling, SMC proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current studies were to examine the potential of LRP1 to modulate vascular physiology under non-atherogenic conditions. Approach and Results We found smLRP1-/- mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not due to excessive PDGF signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1-/- vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1) which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis resulting in fragmentation of elastic fibers. Importantly, our studies discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor (CTGF), an LRP1 ligand and a key mediator of fibrosis. Conclusions Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and CTGF protein levels. These studies highlight two new molecules, CTGF and HtrA1, which contribute to detrimental changes in the vasculature and therefore represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor.

Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.