Adrenomedullin induces matrix metalloproteinase-2 activity in rat aortic adventitial fibroblasts

Background

The delicate balance of the extracellular matrix (ECM) determines the stiffness of the vascular wall, and adventitial fibroblasts are involved in ECM formation by synthesizing and degrading matrix proteins. In the present study, we examined the effect of the bioactive peptide adrenomedullin (AM) on activity and expression of matrix metalloproteinases (MMPs) in cultured aortic adventitial fibroblasts.

Methods and results

In cultured adventitial fibroblasts isolated from aorta of adult Wistar rats, 10⁻⁶ mol/L angiotensin II (Ang II) significantly (p < 0.05) down-regulated MMP-2 activity as determined by in vitro gelatin zymography. In contrast, 10⁻⁷ mol/L synthetic rat AM significantly (p < 0.05) stimulated zymographic MMP-2 activity by 23%, increasing intracellular cAMP, and AM abolished the action of Ang II, augmenting the MMP-2 activity. Similarly, Ang II down-regulated MMP-2 protein expression assessed by Western blotting, whereas AM increased it. Furthermore, 8-bromo-cAMP, an analogue of cAMP, mimicked the effect of AM, and H-89, an inhibitor for protein kinase A (PKA), significantly decreased the basal and AM-induced MMP-2 activity.

Conclusion

This study provides a new insight into the biological action of AM and its intracellular signaling system of cAMP/PKA stimulating the matrix degrading enzyme MMP-2, suggesting an important role for this molecule in modulating ECM deposition in the adventitial layer.     

Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1

Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system

The developing central nervous system is partitioned into compartments by boundary cells, which have different properties than compartment cells, such as forming neuron-free zones, proliferating more slowly and acting as organizing centers. We now report that in mice the bHLH factor Hes1 is persistently expressed at high levels by boundary cells but at variable levels by non-boundary cells. Expression levels of Hes1 display an inverse correlation to those of the proneural bHLH factor Mash1, suggesting that downregulation of Hes1 leads to upregulation of Mash1 in non-boundary regions, whereas persistent and high Hes1 expression constitutively represses Mash1 in boundary regions. In agreement with this notion, in the absence of Hes1 and its related genes Hes3 and Hes5, proneural bHLH genes are ectopically expressed in boundaries, resulting in ectopic neurogenesis and disruption of the organizing centers. Conversely, persistent Hes1 expression in neural progenitors prepared from compartment regions blocks neurogenesis and reduces cell proliferation rates. These results indicate that the mode of Hes1 expression is different between boundary and non-boundary cells, and that persistent and high levels of Hes1 expression constitutively repress proneural bHLH gene expression and reduce cell proliferation rates, thereby forming boundaries that act as the organizing centers.     

TRIM59 interacts with ECSIT and negatively regulates NF-κB and IRF-3/7-mediated signal pathways

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.     

Diel changes in resource use and diet overlap in temperate stream fishes

Interspecific variation in diel-scale temporal niches is common in natural communities. Such variation changes population dynamics via effects on the growth and reproduction of individuals. Also at the community level, theory predicts that animals can reduce competition for shared resources by changing diel activity in certain situations. However, the role of diel activity at the community-level has not been examined sufficiently. In this study, to examine whether the diel-scale temporal niche act as a competition-mitigating mechanism for stream fishes at the community level, we surveyed diel changes in microhabitat use and foraging, and the pattern of interspecific diet overlap in the middle reaches of a temperate stream where various fish species that seemed to be either nocturnal or diurnal coexisted. Our results suggest that the fishes forage during both daytime and night, but change their foraging mode at different times of the day, so that the foraging habits of these fish species cannot be divided simply into nocturnal and diurnal. Furthermore, fishes appeared to aggregate in the vicinity of common food resources during time zones with high availability of the resources, and therefore, inter-guild diet overlap was high during certain time zones. On the other hand, when inter-guild diet overlap was low, each fish species used foods or microhabitats that did not any have the potential to be used by species of another guild. Therefore, we conclude that variation in diel niche use is influenced by variation in the fundamental niche and food supply or availability rather than by competitive interaction between fishes in the stream fish community.     

Ubiquitylation of ε-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the ε-subunit of coatmer complex, ε-COP. PIRH2 promotes the ubiquitylation of ε-COP in vitro and in vivo and consequently promotes the degradation of ε-COP. The interaction between PIRH2 and ε-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of ε-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.