HIF-1α is a normally labile proangiogenic transcription factor that is stabilized and activated in hypoxia. Although the von Hippel Lindau (VHL) gene product, the ubiquitin ligase responsible for regulating HIF-1α protein levels, efficiently targets HIF-1α for rapid proteasome-dependent degradation under normoxia, HIF-1α is resistant to the destabilizing effects of VHL under hypoxia. HIF-1α also associates with the molecular chaperone Hsp90. To examine the role of Hsp90 in HIF-1α function, we used renal carcinoma cell (RCC) lines that lack functional VHL and express stable HIF-1α protein under normoxia. Geldanamycin (GA), an Hsp90 antagonist, promoted efficient ubiquitination and proteasome-mediated degradation of HIF-1α in RCC in both normoxia and hypoxia. Furthermore, HIF-1α point mutations that block VHL association did not protect HIF-1α from GA-induced destabilization. Hsp90 antagonists also inhibited HIF-1α transcriptional activity and dramatically reduced both hypoxia-induced accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. These findings demonstrate that disruption of Hsp90 function 1) promotes HIF-1α degradation via a novel, oxygen-independent E3 ubiquitin ligase and 2) diminishes HIF-1α transcriptional activity. Existence of an Hsp90-dependent pathway for elimination of HIF-1α predicts that Hsp90 antagonists may be hypoxic cell sensitizers and possess antiangiogenic activityin vivo, thus extending the utility of these drugs as therapeutic anticancer agents.

The early progenitor cells to the pancreatic islets in the mouse have been characterized so as to re-examine their possible lineage relationships to the four islet cell types found in mature islets. Insulin and glucagon were both first expressed at embryonic day 9.5, and many cells co-expressed these two markers, as shown by light and electron microscopic analysis using double-label immunohistochemistry. Incubation of embryonic pancreas with 1% glutaraldehyde, a fixative commonly used by electron microscopists, abolished this reactivity, thereby explaining reported difficulties in detecting these precursor cells. Using antisera specific for neu-ropeptide Y (NPY) a peptide with considerable homology to pancreatic polypeptide (PP), we show that NPY first appears with insulin and glucagon immunore-activity at E9.5, and is co-expressed with glucagon in a majority of adult cells. As we have previously reported, PP itself is first detectable immunocytochemically at postnatal day 1 with PP-specific antibodies. However, antibodies raised against bovine PP are shown by dot blotting to recognize NPY with comparable avidity, indi-cating that a recent report of islet progenitor cells containing PP at E9.5 (Herrera, P. L., Huarte, J., Sanvito, F., Meda, P., Orci, L. and Vassalli, J. D. (1991) Devel-opment 113, 1257-1265), actually represents cross-reactivity to NPY. The data support a model in which early precursor cells to the endocrine pancreas co-activate and co-express a set of islet cell hormone and neural genes, whose expression is both selectively increased and extinguished as development proceeds, concomitant with a restriction to the patterns of expression characteristic of mature islet cell types.

Although adrenomedullin (AM) previously has been identified in human tumors, its role has remained elusive. Analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed AM mRNA in 18 of 20 human normal tissues representing major organs, and 55 of 58 (95%) malignant cell lines. Western blot and high performance liquid chromatography analysis showed immunoreactive AM species of 18, 14, and 6 kDa that are consistent with the precursor, intermediate product, and active peptide, respectively. Immunohistochemistry and in situ RT-PCR performed on paraffin-embedded tumor cell lines of various tissue origins exhibited AM cytoplasmic staining. Neutralizing monoclonal antibody to AM inhibits tumor cell growth in a concentration-dependent manner, an effect that was reversed with the addition of exogenous AM. Responding tumor cells were shown to have approximately 50,000 AM receptors per cell by Scatchard analysis with 125I-AM and expressed AM receptor mRNA by RT-PCR. Our data showed 36 of 48 (75%) tumor cell lines expressed AM receptor mRNA by RT-PCR assessment, all of them also expressed AM. In the presence of AM, cAMP levels were shown to increase in tumor cells. Our collective data demonstrate that AM and AM receptor are expressed in numerous human cancer cell lines of diverse origin and constitute a potential autocrine growth mechanism that could drive neoplastic proliferation.

Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress–induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress–induced release of adrenomedullin, which in turn activated its G_s-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This G_s/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gα_s showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress–induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent G_s-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.

Bone is a common site of cancer metastasis. Breast, prostate, and lung cancers show a predilection to metastasize to bone. Recently, we reported that the chemokine interleukin 8 (IL-8) stimulates both human osteoclast formation and bone resorption. IL-8 mRNA expression was surveyed in a panel of human breast cancer lines MDA-MET, MDA-MB-231, MDA-MB-435, MCF-7, T47D, and ZR-75, and the human lung adenocarcinoma cell line A549. IL-8 mRNA expression was higher in cell lines with higher osteolytic potential in vivo. Human osteoclast formation was increased by MDA-MET or A549 cell-conditioned medium, but not by MDA-MB-231. Pharmacologic doses of receptor activator of nuclear factor-κB (RANK)-Fc or osteoprotogerin had no effect on the pro-osteoclastogenic activity of the conditioned medium; however, osteoclast formation stimulated by conditioned medium was inhibited 60% by an IL-8-specific neutralizing antibody. The data support a model in which tumor cells cause osteolytic bone destruction independently of the RANK ligand (RANKL) pathway. Tumor-produced IL-8 is a major contributor to this process. The role of secreted IL-8 isoforms was examined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, which detected distinct IL-8 isoforms secreted by MDA-MET and MDA-231 cells, suggesting different pro-osteoclastogenic activities of the two IL-8-derived peptides. These data indicate that (a) osteoclast formation induced by MDA-MET breast cancer cells and A549 adenocarcinoma cells is primarily mediated by IL-8, (b) cell-specific isoforms of IL-8 with distinct osteoclastogenic activities are produced by tumor cells, and (c) tumor cells that support osteoclast formation independent of RANKL secrete other pro-osteoclastogenic factors in addition to IL-8.

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.