Expression of CCAAT/enhancer binding proteins alpha and beta in the porcine ovary and regulation in primary cultures of granulosa cells

CCAAT/enhancer binding proteins alpha and beta (CEBPA/ CEBPB) were evaluated in the porcine ovary during the estrous cycle. CEBPB mRNA was present in antral follicles and was significantly increased in healthy corpora lutea (CL), whereas CEBPA mRNA was constitutively expressed in these structures. Both isoforms of CEBPA (42 and 30 kDa) exhibited greater expression in preovulatory follicles, and the 42-kDa isoform increased in CL, whereas the 30-kDa isoform decreased. All major isoforms of CEBPB (38, 34, and 20 kDa) were expressed, with the 34- and 20-kDa isoforms being more abundant in preovulatory follicles and further increased in CL. The effects of FSH and cAMP analogue on the distribution of CEBP isoforms were evaluated in primary cultures of porcine granulosa cells. FSH and 8-Br-cAMP had little stimulatory effect on isoform distribution, but cAMP treatment for 24 h tended to decrease the 30-kDa form of CEBPA and the 34-kDa form of CEBPB. The 34-kDa form of CEBPB was decreased by the protein kinase A inhibitor H89 at 4 h (with FSH treatment), and by both protein kinase A and phosphatidylinositol 3-kinase inhibitors at 24 h of treatment. In transfected granulosa cells, FSH and cAMP analogue stimulated a CEBP consensus sequence-reporter construct that was blocked by H89. These data implicate protein kinase A as the major regulator of CEBPB isoform distribution and CEBP-mediated transactivation in granulosa cells. The differential expression of specific CEBPA/B isoforms observed in maturing follicles and CL may contribute to changes in follicular cell differentiation and increasing steroidogenic capacity.     

Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from RAGE^−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope. This work was supported by NIH grants R01 GM37631 and GM68481.     

Isolation and partial characterization of lumican and decorin from adult chicken corneas. A keratan sulfate-containing isoform of decorin is developmentally regulated.

The proteoglycans extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.     

A structurally variant form of the 2B4 antigen is expressed on the cell surface of mouse mast cells

2B4 (CD244) is a 66-kDa CD2 family protein expressed on natural killer (NK) cells. Mouse NK cells express two isoforms of 2B4, termed 2B4L and 2B4S, whose molecular masses are 42 kDa and 36 kDa, respectively. In this study, we biochemically characterize the 2B4 antigen that was newly found on mouse bone marrow-derived mast cells (BMMC). Anti-2B4 mAb immunoprecipitated glycoproteins with a molecular mass of 60 kDa from BMMC. Removal of N-linked sugars from the antigen by N-glycosidase F treatment yielded two protein backbones of 35 kDa and 25 kDa, indicating that BMMC express the 2B4S isoform, but not 2B4L. Nucleotide sequence analyses confirmed that BMMC transcribe 2B4S mRNA. The preferential expression of the 2B4S isoform and the detection of an additional 25-kDa glycoprotein on BMMC indicate that differences in the structure of 2B4 antigen exist between BMMC and NK cells.     

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.     

Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis

Arabidopsis Rubisco activase was recently shown to be regulated by redox changes in the larger (46-kDa) isoform specifically mediated by thioredoxin-f [Zhang and Portis (1999) Proc Natl Acad Sci USA 96: 9438–9443]. Reduction greatly increases the activity of the 46-kDa isoform and the native protein at physiological ATP/ADP ratios. In this study we conducted additional experiments to characterize the regulation of Rubisco activase by thioredoxin-f. The K_m for both ATP hydrolysis and Rubisco activation by the 46-kDa isoform was lowered by 4 to 5-fold after reduction, but the maximum activity was increased by only 10%. Only 0.35 μM thioredoxin-f was required for a half-maximal activity change after a 10 min preincubation and activation with 1 μM was complete after 10 min. Equal amounts of 46-kDa and 43-kDa isoforms were required for a complete inhibition of the Rubisco activation activity after a reduction-oxidation cycle and assay at an ATP/ADP ratio of 3:1, whereas activity was only inhibited by 50% at a 2:1 ratio (43-/46-kDa) of the isoforms. This requirement is consistent with the fact that Arabidopsis normally contains about a 1:1 ratio of the two isoforms at both the mRNA and protein levels. Redox titrations indicated a midpoint potential of −344 mV for the 46-kDa isoform as compared to −342 mV for spinach fructose 1,6-bisphosphatase at pH 7.9, consistent with previous reports indicating that these proteins are co-regulated by light intensity in a similar manner. This revised version was published online in June 2006 with corrections to the Cover Date.