Isoform-specific regulation of platelet-derived growth factor activity and binding in osteoblast-enriched cultures from fetal rat bone.

In osteoblast-enriched cultures from fetal rat bone, the A-chain homodimer of platelet-derived growth factor (PDGF-AA) is less potent than the PDGF isoforms containing B chain subunits (PDGF-AB and PDGF-BB), but normal osteoblasts appear to synthesize only PDGF-A subunit mRNA and polypeptide. However, other agents may regulate PDGF-AA activity in skeletal tissue. Pretreatment of osteoblast-enriched cultures with interleukin 1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) synergistically enhanced the mitogenic effect of PDGF-AA coincident with increased binding site occupancy, but neither factor augmented PDGF-BB activity or binding. Polyacrylamide gel analysis showed 125I-PDGF-AA binding complexes predominantly at greater than 200 kD and faint labeling at 185 kD. After IL-1 alpha or TNF-alpha pretreatment, PDGF-AA binding increased at both sites, but this effect was more striking at 185 kD, which co-migrated with 125I-PDGF-BB-labeled complexes. PDGF-AA binding sites were rapidly lost by comparison to those for PDGF-BB in cycloheximide-treated cultures, but they remained relatively enhanced by IL-1 alpha and TNF-alpha pretreatment. These studies indicate that IL-alpha and TNF-alpha increase PDGF-AA binding and activity for osteoblasts by mechanisms that are at least in part independent of new receptor synthesis, and suggest regulatory events that could control how PDGF binding sites specifically recognize different ligands.

Pharmacokinetic evaluation of two human epidermal growth factors (hEGF51 and hEGF53) in rats.

The pharmacokinetic profiles of two iodinated human epidermal growth factors (125I-hEGF51 and 125I-hEGF53) in rats receiving a single intravenous dose have been investigated using three independent bioanalytical techniques. Based on a tetrachloroacetic acid precipitation methodology, hEGF51 and hEGF53 were found to have distribution half-lives of 0.80 +/- 0.2 and 0.80 +/- 0.18 min, and elimination half-lives of 79.8 +/- 24.1 and 77.9 +/- 21.1 min, respectively. Evaluated by immunoprecipitation, distribution half-lives were 0.59 +/- 0.09 and 0.63 +/- 0.15 min, and elimination half-lives were 117.8 +/- 22.9 and 118.7 +/- 38.8 min, respectively. Both precipitation techniques produced similar, parallel plasma concentration-time curves, and there were no significant differences in other calculated kinetic parameters, including clearance and volume of distribution evaluated by either technique. Plasma disposition profiles of both peptides were also confirmed by visualization with SDS-PAGE and autoradiography, and were found to be similar to those generated by tetrachloroacetic acid and immunoprecipitation methods. Thus, three independent methods strongly suggest that both peptides have the same disposition profile, which exhibits a very rapid disappearance rate in the distribution phase followed by a much slower elimination process. These results also indicate that the pharmacokinetic behavior of human epidermal growth factor is not altered by deletion of two amino acids from the carboxyl terminus. In addition, the incubation study suggests that about 23% of the exogenous peptides were associated with red blood cells.

Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone.

Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.

Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor.

Monoclonal antibodies to the rat hepatic glucocorticoid receptor (GR) were produced by using 4000-fold-purified unactivated rat hepatic GR as the immunogen in an immunization in vitro. Hybridomas were screened for anti-GR antibody production by using an enzyme-linked immunosorbent assay. The antibody, 3A6, described here, is an IgM (lambda). The interaction of 3A6 with the purified GR was explored by sedimentation analysis, where a shift of the 9 S GR to a form with a higher s20,w value was demonstrated. Binding specificity and sensitivity were demonstrated by protein immunoblotting. 3A6 cross-reacted with all rat tissue glucocorticoid receptors (GRs) examined, except those of the brain. Species cross-reactivity was observed with other mammalian GRs (from human CEM-C7 cells and from pig and mouse liver). Immunocytochemical localization of the GR was assessed by indirect immunofluorescence in intact fixed cells, which demonstrated intense cytoplasmic staining in the absence of pretreatment with glucocorticoids and nuclear localization when cells were pretreated with glucocorticoids. This monoclonal antibody significantly inhibited steroid binding to unoccupied receptor and DNA binding of activated steroid-receptor complexes. Furthermore, preincubation of the purified activated GR complex with 3A6 prevented phosphorylation of the GR in vitro. Thus 3A6 differs from previous monoclonal antibodies to the GR in its capacity to cross-react with the human GR and by its specificity for an epitope on or near a functional domain of the GR.

Human myometrium: a new potential source of prolactin.

Human myometrium is shown for the first time to produce prolactin in vitro. This prolactin is similar to pituitary prolactin by criteria of immunologic identity, gel chromatography and bioassay. The de novo synthesis of myometrical prolactin is supported by no detectable prolactin in initial tissue homogenate, nondetectable prolactin production during the first 24 hours of culture, cycloheximide inhibition of prolactin production with recovery of production in control medium, and tritiated leucine incorporation into prolactin. Although human myometrium is capable of producing prolactin without the addition of exogenous hormones, the addition of estrogen and progesterone, respectively, enhances and suppresses prolactin production in contrast to decidualized human endometrium where opposite effects on prolactin production are found.

Stimulatory activity of serum on prolactin production by human decidua.

Fetal calf serum (FCS) enhances the synthesis and secretion of prolactin (PRL) by explants of human decidua incubated in Gey's balanced salt solution. In order to further characterize the factors involved in the prolactin-stimulating activity (PSA) of serum, decidua was incubated in Gey's buffer alone and in buffer containing either FCS, bovine serum albumin (BSA), or human serum (HS). When buffer was supplemented with varying concentrations of FCS, ad dose-related PSA was apparent between 0.1% and 10%. The PSA of FCS was not altered by eithr heating (85degrees C for 30 minutes), boiling, or dialysis. The protein fraction of FCS, precipitated with trichloroacetic acid, redissolved in dilute NaOH, and dialyzed against Dulbecco's phosphate buffer, demonstrated PSA comparable to that of whole serum. A similar concentration of BSA added to Gey's buffer did not stimulate the production of PRL by decidua. The PSA of human serum obtained from euprolactinemic men and women was not different from that of FCS. These data indicate that FCS and HS contain a substance(s) (1) which stimulates the secretion of PRL by human decidua, (2) which is very stable, being resistant to dialysis, boiling, and denaturation by trichloroacetic acid, (3) and which is most likely a large polypeptide with a molecular weight greater than 12,000 or a smaller moiety tightly bound to a protein.

Evidence for a nonpituitary source of amniotic fluid prolactin.

Prolactin levels were determined during a gonadotropin-induced pregnancy following hypophysectomy for a chromophobe adenoma. Maternal plasma prolactin concentrations did not vary significantly from prepregnancy values throughout gestation, remaining between 25 and 35 ng/ml. Fetal prolactin levels were 55 ng/ml and maternal levels were 29 ng/ml at delivery. Amniotic fluid prolactin concentration was approximately 100 ng/ml. Decidual tissue isolated from the maternal surface of the chorion released significant amounts of prolactin into the medium during a 24-hour incubation. Final concentrations of prolactin in the incubation medium were as high as 196 ng/ml. It is concluded that after hypophysectomy (1) prolactin is present in the maternal circulation during pregnancy, and the concentration does not change significantly throughout gestation; (2) fetal and amniotic fluid prolactin concentrations near term do not differ significantly from those reported for normal pregnancy; and (3) the capacity of the decidua to release prolactin in vitro is not diminished compared with normal term decidua, suggesting a nonpituitary source of amniotic fluid prolactin.

Decidua: a possible source of amniotic fluid prolactin.

The source of amniotic fluid prolactin was investigated with the use of amnion, chorion, placenta, and decidual tissue taken from term human pregnancy. Decidua alone of these tissues contained significant quantities of prolactin. The release of decidual prolactin was affected by the presence or absence of oxygen and protein, and the amount of prolactin released far exceeded the decrease in tissue content during incubation. It is concluded that: (1) decidua may be a major source of amniotic fluid prolactin, (2) synthesis of prolactin occurs during incubation of decidua, and (3) sufficient prolactin is present in the decidua to account for that found in amniotic fluid at term.