Delta Protein Levels Research Articles

Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney.

Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.

Notch and delta mRNAs in early‐stage and mid‐stage drosophila embryos exhibit complementary patterns of protein‐producing potentials

Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3' processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early- and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3' UTR and mRNA 3' processing factors.

Analysis of protein kinase C delta (PKC δ) expression in endometrial tumors

Endometrial cancer is the most common gynecologic malignancy in the United States. However, its underlying molecular mechanisms are poorly understood; and few prognostic indicators have been identified. The protein kinase C (PKC) family has been shown to regulate pathways critical to malignant transformation; and in endometrial tumors, changes in PKC expression and activity have been linked to a more aggressive phenotype and poor prognosis. We have recently shown that PKC delta is a critical regulator of apoptosis and cell survival in endometrial cancer cells; however, PKC delta levels in endometrial tumors had not been determined. We used immunohistochemistry to examine PKC delta protein levels in normal endometrium and endometrioid carcinomas of increasing grade. Normal endometrium exhibited abundant nuclear and cytoplasmic staining of PKC delta confined to glandular epithelium. In endometrial tumors, decreased PKC delta expression, both in intensity and fraction of epithelial cells stained, was observed with increasing tumor grade, with PKC delta being preferentially lost from the nucleus. Consistent with these observations, endometrial cancer cell lines derived from poorly differentiated tumors exhibited reduced PKC delta levels relative to well-differentiated lines. Treatment of endometrial cancer cells with etoposide resulted in a translocation of PKC delta from cytoplasm to nucleus concomitant with induction of apoptosis. Decreased PKC delta expression, particularly in the nucleus, may compromise the ability of cells to undergo apoptosis, perhaps conferring resistance to chemotherapy. Our results indicate that loss of PKC delta is an indicator of endometrial malignancy and increasing grade of cancer. Thus, PKC delta may function as a tumor suppressor in endometrial cancer.

C/EBP DNA-binding activity is upregulated by a glucocorticoid-dependent mechanism in septic muscle.

Sepsis-induced muscle cachexia is associated with increased expression of several genes in the ubiquitin-proteasome proteolytic pathway, but little is known about the activation of transcription factors in skeletal muscle during sepsis. We tested the hypothesis that sepsis upregulates the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and -delta in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture, and control rats were sham operated. C/EBP-beta and -delta DNA-binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In addition, C/EBP-beta and -delta nuclear protein levels were determined by Western blot analysis. Sepsis resulted in increased DNA-binding activity of C/EBP, and supershift analysis suggested that this reflected activation of the beta- and delta-isoforms of C/EBP. Concomitantly, C/EBP-beta and -delta protein levels were increased in the nuclear fraction of skeletal muscle. In additional experiments, we tested the role of glucocorticoids in sepsis-induced activation of C/EBP-beta and -delta by treating rats with the glucocorticoid receptor antagonist RU-38486. This treatment inhibited the sepsis-induced activation of C/EBP-beta and -delta, suggesting that glucocorticoids participate in the upregulation of C/EBP in skeletal muscle during sepsis. The present results suggest that C/EBP-beta and -delta are activated in skeletal muscle during sepsis and that this response is, at least in part, regulated by glucocorticoids.

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

CCAAT/enhancer binding protein isoforms expression in the colon of neonatal mice.

Development of murine proximal colon follows a complex pattern of morphological and functional differentiation. Molecular mechanisms and factors responsible for colon-specific gene expression remain to be established. In an attempt to identify some of these factors, we examined the expression of the alpha, beta, and delta isoforms of the CCAAT/enhancer binding protein (C/EBP) transcription factor gene family during murine colon development. Whereas C/EBP alpha mRNA levels are reduced during the third post-natal week, C/EBP alpha 42 and 30 kD proteins levels decrease between post-natal days 8 and 21. C/EBP beta mRNA levels increase between post-natal days 4 and 8 and remain constant subsequently, in contrast to a decrease in C/EBP beta protein levels between post-natal days 11 and 15. C/EBP delta mRNA levels increase gradually while C/EBP delta protein levels show variations during post-natal development. Changes in C/EBP DNA binding activity coincides with modifications in C/EBP isoforms expression. By indirect immunofluorescence, we show restriction of C/EBP alpha expression to differentiated surface epithelial cells during crypt formation. C/EBP alpha is predominantly nuclear with some cytoplasmic staining at all developmental stages. C/EBP beta and delta are both predominantly nuclear in crypt and differentiated surface epithelial cells, as well as in various cells of the lamina propria and muscular layers. Thus, specific C/EBP isoforms are differentially regulated during murine colon post-natal development. Differential C/EBP isoforms pattern of expression suggests a role for these transcription factors in colon-specific gene expression during development.