Fibroblast growth factor: effects on osteogenesis and chondrogenesis in the chick embryo.

In vivo effects of fibroblast growth factor (FGF) on osteogenesis were evaluated in the chick embryo. Autoradiographic studies of 3H-proline labeling over bone matrix indicated that 24 h after treatment on day 11, FGF stimulated osteogenic cell proliferation, while inhibiting the production of bone matrix collagen. However, 4 days after multiple doses of FGF, the large pool of newly formed osteogenic and chondrogenic cells expressed their function with the increased formation of matrix. The data provide in vivo evidence of the effects of exogenous FGF on osteogenesis and also point to a possible role for FGF both in embryonic osteogenesis and in fracture repair.     

Long and short mRnas transcribed from the medaka fish transposon Tol2 respectively exert positive and negative effects on excision

The medaka fish transposable element, Tol2, is a member of the hAT family of transposons. It has been directly demonstrated to be active and two mRNAs, differing in length, have been isolated. They cover exons 1-4 and exons 2-4 and the longer form has already been proven to catalyse transposition reactions. However, the function of the shorter mRNA in medaka cells has hitherto remained unclear. In the present study, first we constructed a quantitative system to detect Tol2 excision using an indicator plasmid carrying a non-autonomous Tol2 within its lacZ gene; second we injected mRNAs with the plasmid into medaka eggs. Excision of Tol2 was detected as E. coli blue colonies caused by the recovery of lacZ activity. Addition of the longer mRNA increased excision, but the shorter did not. Moreover, co-injection of both mRNAs greatly lowered the frequency compared with the case of treatment with the longer mRNA alone. These results indicate that the shorter mRNA has an inhibitory effect on the excision reaction, and that the N-terminal region of the transposase encoded by exon 1, including a BED zinc finger, presumably plays an important role in excision. Here, we suggest a regulatory mechanism of Tol2 transposition involving the expression of these mRNAs.     

Host plant nitrogen enrichment has both positive and negative effects on the larval growth of a specialist butterfly

1. The nitrogen limitation hypothesis posits that phytophagous insects benefit from nitrogen enrichment of their host plants through a reduction of the concentration of toxic compounds and an increase of free amino acids and proteins. However, species' response to nitrogen enrichment varies substantially and high nitrogen levels are associated with population decline, suggesting there are major costs to feeding on nitrogen‐rich host plants. 2. To test the hypothesis that larval growth performance is maximal at intermediate nitrogen enrichment, nitrogen levels were measured in 18 populations of the host plant of Lycaena helle, a specialist butterfly inhabiting nutrient‐poor wet meadows. The nitrogen content of host plants was then modified to mirror average natural nitrogen levels (C), highest field‐recorded levels (T1), and levels higher than those observed across our study populations (T2). 3. Caterpillars fed with T1 leaves had a greater maximum body mass than caterpillars of the C group because of their improved food assimilation during the early stages of their development. Caterpillars of C and T2 groups had similar growth patterns but high nitrogen content had detrimental effects, as caterpillars fed with T2 leaves had a slower ingestion rate than C and T1 groups. 4. Quantifying the fitness consequences of these changes in growth performance is necessary to fully understand the implications of nitrogen enrichment for L. helle (rapid growth may result in fitness costs). However, conservation plans for this emblematic glacial relict species should also consider the preservation of its host plant quality to ensure its persistence.     

Belowground positive and negative feedbacks on CO2 growth enhancement

In this paper we present a conceptual model of integrated plant-soil interactions which illustrates the importance of identifying the primary belowground feedbacks, both positive and negative, which can simultaneously affect plant growth responses to elevated CO₂. The primary negative feedbacks share the common feature of reducing the amount of nutrients available to plants. These negative feedbacks include increased litter C/N ratios, and therefore reduced mineralization rates, increased immobilization of available nutrients by a larger soil microbial pool, and increased storage of nutrients in plant biomass and detritus due to increases in net primary productivity (NPP). Most of the primary positive feedbacks share the common feature of being plant mediated feedbacks, the only exception being Zak et al.'s hypothesis that increased microbial biomass will be accompanied by increased mineralization rates. Plant nutrient uptake may be increased through alterations in root architecture, physiology, or mycorrhizal symbioses. Further, the increased C/N ratios of plant tissue mean that a given level of NPP can be achieved with a smaller supply of nitrogen.Identification of the net plant-soil feedbacks to enhanced productivity with elevated CO₂ are a critical first step for any ecosystem. It is necessary, however, that we first identify how universally applicable the results are from one study of one ecosystem before ecosystem models incorporate this information. The effect of elevated CO₂ on plant growth (including NPP, tissue quality, root architecture, mycorrhizal symbioses) can vary greatly for different species and environmental conditions. Therefore it is reasonable to expect that different ecosystems will show different patterns of interacting positive and negative feedbacks within the plant-soil system. This inter-ecosystem variability in the potential for long-term growth responses to rising CO₂ levels implies that we need to parameterize mechanistic models of the impact of elevated CO₂ on ecosystem productivity using a detailed understanding of each ecosystem of interest.     

In vitro effects of glucose and growth factors on limb bud and mandibular arch chondrocytes maintained at various serum concentrations

In human and experimental diabetic pregnancy there is an increased risk of congenital malformation in the offspring. Some malformations involve growth retardation and altered chondrocyte differentiation, suggesting that a diabetic milieu may modify embryonic cell replication and the development of (pre)chondrocytes. The aim of the present work was to study the effects of a diabetes-like environment in vitro on the growth and differentiation of rat chondrocytes in the presence of specific growth factors and different concentrations of serum. This was performed with a modified micromass culture system of embryonic (pre)chondrocytes from the limb bud and mandibular arch areas using medium supplemented with different glucose concentrations and with serum from diabetic rats. An elevated ambient glucose concentration inhibited the growth of mature chondrocytes in vitro, and this effect was diminished in a serum-rich culture milieu. The (pre)chondrocytes exhibited a marked dependence on the serum level in the culture medium for optimal in vitro development. Diabetic rat serum had the lowest stimulatory capacity of the three different types tested (at similar glucose concentrations), suggesting a deficiency of growth-stimulating factor(s) rather than the presence of inhibiting factor(s) in this type of serum. One of the deficient factor(s) in diabetic rat serum may be similar to IGF-II, but a combined deficiency of several growth-stimulating agents is likely to be present. Chondrocytes originating from the mandibular arch in general appeared more sensitive to MSA and IGF-II than those from the limb buds. The present observations support the notion that while diabetes-induced hyperglycemia in the conceptus contributes to severe growth retardation of the mandibular arch, additional factors also play a role.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

Insulin-like growth factor (IGF) binding protein-4 is both a positive and negative regulator of IGF activity in vivo

IGFs are required for normal prenatal and postnatal growth. Although actions of IGFs can be modulated by a family of IGF-binding proteins (IGFBPs) in vitro, these studies have identified a complicated pattern of stimulatory and inhibitory IGFBP effects, so that understanding relevant aspects of IGFBP action in vivo has been limited. Here we have produced a null mutation of one specific IGFBP, IGFBP-4, which is coexpressed with IGF-II early in development. Surprisingly, mutation of IGFBP-4, believed from in vitro studies to be exclusively inhibitory, leads to a prenatal growth deficit that is apparent from the time that the IGF-II growth deficit first arises, which strongly suggests that IGFBP-4 is required for optimal IGF-II-promoted growth during fetal development. Mice encoding a mutant IGFBP-4 protease (pregnancy-associated plasma protein-A), which facilitates IGF-II release from an inactive IGF-II/IGFBP-4 complex in vitro, are even smaller than IGFBP-4 mutant mice. However, the more modest IGFBP-4 growth deficit is completely restored in double IGFBP-4/pregnancy-associated plasma protein-A-deficient mice. Taken together these results indicate not only that IGFBP-4 functions as a local reservoir to optimize IGF-II actions needed for normal embryogenesis, but also establish that IGFBP-4 proteolysis is required to activate most, if not all, IGF-II mediated growth-promoting activity.     

Fibroblast growth factor homologous factors and the islet brain-2 scaffold protein regulate activation of a stress-activated protein kinase

Fibroblast growth factor homologous factors (FHFs) form native intracellular complexes with the mitogen-activated protein kinase (MAPK) scaffold protein islet-brain 2 (IB2) in adult brain. FHF binding to IB2 facilitates recruitment of the MAPK p38delta (SAPK4), while failing to stimulate binding of JNK, the preferred kinase of the related scaffold IB1 (JIP-1). We now report further biochemical evidence supporting FHFs as regulators of IB2 scaffold activity. Mixed lineage kinase 3 (MLK3) and IB2 synergistically activate p38delta but not the MAPKs JNK-1 and p38alpha. Binding of p38delta to IB2 is mediated by the carboxyl-terminal half of the scaffold (IB2(Delta1-436)). FHF2 also binds weakly to IB2(Delta1-436) and can thereby increase p38delta interaction with IB2(Delta1-436). FHF-induced recruitment of p38delta to IB2 is accompanied by increased levels of activated p38delta, and synergistic activation of p38delta by MLK3 and IB2 is further enhanced by FHF2. Consistent with a role for FHFs as signaling molecules, FHF2 isolated from rat brain is serine/threonine-phosphorylated, and FHF can serve as a substrate for p38delta in vitro. These results support the existence of a signaling module in which IB2 scaffolds a MLK3/MKK/p38delta kinase cascade. FHFs aid in recruitment of p38 to IB2 and may serve as kinase substrates.     

Gastrointestinal growth factors and hormones have divergent effects on Akt activation

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.     

Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, was not affected in mice deficient for the IIIb isoform. FgfR2-IIIb(-/-) (lac)(Z) mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence did not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but did prevent induction of Shh and Fgf4, indicating that they are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. We propose that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth.     

Fibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4

The mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1-FGFR4 and FGF2-FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2-FGFR4 but not by FGF1-FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner.     

Comparative effects of cytosine arabinoside and a prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme

The present study was designed to compare effects of an established inhibitor of cell proliferation and growth, cytosine arabinoside (Ara C), with that of a prostaglandin E2 (PGE2) antagonist, AH6809, on chondrogenesis in cultured mesenchyme derived from stage 25 chick limb buds. Continuous treatment of cell cultures with 10(-4) M AH6809 prevented completely the twofold increases in DNA content of control cultures which occurred between Day 1 and Day 5 of culture and also produced 90% inhibition of chondrogenesis occurring in control cultures during this same period. Treatment of cells with Ara C (0.1-0.5 microgram/ml) produced equivalent inhibition of DNA content during the same time period; however, chondrogenesis, as evaluated on Day 5 of cell culture, remained at approximately 90% of control cultures. These results indicate that the inhibitory effect of PGE2 receptor blockade on cell growth in these cultures cannot account for the potent inhibitory effects observed on differentiation of cartilage and provide further evidence in support of the notion that PGE2 plays an important initiating role in the process of chondrocyte differentiation within limb mesenchyme.     

The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.     

Signal transduction in T helper cells: CD4 coreceptors exert complex regulatory effects on T cell activation and function

The immune system provides a highly sophisticated surveillance mechanism to detect diverse antigens and to protect the host organism from invading pathogens and altered cells (e.g., virus-infected and tumor cells). Adaptive immune responses depend on the recognition of antigen by specific antigen receptors that are expressed on the surface of T and B lymphocytes. Helper T cells provide regulatory functions and direct the adaptive immune system to respond appropriately to a particular antigen (i.e., cytotoxic T cell responses against viral infections and tumor cells, humoral responses against extracellular bacteria and parasitic worms). Helper T cells express CD4 coreceptors, which recognize conserved domains on proteins expressed by the class II major histocompatibility complex, the same proteins that present antigen to the T cell receptor. Recent progress in T cell biology has identified multiple regulatory functions of CD4 during thymocyte development and antigen stimulation of mature T helper cells. Signaling pathways induced by engagement of CD4 independently of T cell receptor signaling mediate these regulatory functions. In this review, we discuss the regulation of T cell signaling and emphasize the functional consequences of proper and improper CD4 coreceptor signaling.     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.     

Prolactin and transforming growth factor-beta signaling exert opposing effects on mammary gland morphogenesis, involution, and the Akt-forkhead pathway

Both prolactin (PRL) and TGF-beta regulate cell survival in mammary epithelial cells, but their mechanisms of interactions are not known. In primary mammary epithelial cells and the HC11 mouse mammary epithelial cell line, PRL prevented TGF-beta-induced apoptosis, as measured by terminal deoxynucleotidyltransferase dUTP nick-end labeling staining and caspase-3 activation. This effect depended on phosphatidyl inositol triphosphate kinase (PI3K). PI3K activates a downstream serine/threonine kinase, Akt; therefore, we investigated the role of Akt in the interaction between PRL and TGF-beta signaling. Akt activity was inhibited by TGF-beta over a 20- to 60-min time course. In TGF-beta-treated cells, PRL disinhibited Akt in a PI3K-dependent manner. Expression of dominant negative Akt blocked the protective effect of PRL in TGF-beta-induced apoptosis. Transgenic mice overexpressing a dominant-negative TGF-beta type II receptor (DNIIR) in the mammary epithelium undergo hyperplastic alveolar development, and this effect was PRL dependent. Involution in response to teat sealing was slowed by overexpression of DNIIR; furthermore, Akt and forkhead phosphorylation increased in the sealed mammary glands of DNIIR mice. Thus, Akt appears to be an essential component of the interaction between PRL and TGF-beta signaling in mammary epithelial cells both in vitro and in vivo.