Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland

We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal- epithelial interactions mediated by HGF and NRG.     

Polyamines and tumor cells: effect of transformation of chick embryo fibroblasts by Rous sarcoma virus on polyamine levels

The effect of transformation of chick embryo fibroblasts, by Rous sarcoma virus, on intracellular polyamine levels has been studied. A good correlation between spermidine and cellular protein content has been demonstrated. Upon changing the medium, a sharp increase in spermidine level was noticed both in normal and transformed cells. This increase was accompanied by enhanced protein synthesis. The intracellular concentrations of spermine and spermidine were very similar in normal and transformed cells. On the other hand, significant differences in putrescine levels were demonstrated: in normal cultures the intracellular concentration of putrescine reached a plateau approximately 6 days after seeding, whereas a continuous rise of the diamine in transformed cells was noticed. These differences, which were observed in cultured cells, may explain the known accumulation of polyamines during neoplastic growth.     

Glycosidases during chick embryo lung development and their colocalization with proteoglycans and growth factors

During development, the epithelial component of the lung goes through a complex orderly process of branching, following strict patterns of space and time. Proteoglycans, glycosaminoglycans and growth factors are fundamental components of the extracellular matrix and perform a key role in differentiative processes. The embryonic chick lung shows a specific glycosaminoglycan composition at different levels of branching and at different embryonic stages. Proteoglycan and glycosaminoglycan accumulation is the result of secretion, absorption and degradation processes. In this pathway, enzymes, such as glycosidases, growth factors and cytokines are involved. We examined the behaviour of glycosidases, such as beta-hexosaminidases (beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase), beta-glucuronidase and beta-galactosidase, during the development of the lung bud. Our data show that the activity of the enzymes is closely linked to the processes of epithelial proliferation, bronchial tubule lengthening and infiltration of the surrounding mesenchyme. The glycosaminoglycans colocalize with transforming growth factor beta2 and interleukin-1 in the basement membrane and in the mesenchymal areas where the epithelium grows, and are complementary to the presence of the glycosidases. In conclusion, the activity of these glycosidases is spatially and temporally programmed and favors the release of the factors and the events which they influence.     

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.Abbreviations PA Polyamines - PCA Perchloric acid - PUT Putrescine - SPD Spermidine - SPM Spermine     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

Changes in polyamine synthesis and concentrations during chick embryo development

We have measured the activities of the two rate controlling enzymes in polyamine synthesis, L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMDC), and the concentrations of the polyamines, putrescine, spermidine and spermine, in the developing chick embryo from laying to hatching. The embryo exhibited major peaks in the ODC and SAMDC activities as well as in the concentrations of all three polyamines at 15 h (gastrulation), 23-30 h (early organogenesis), days 4-5 (mid-organogenesis), and days 12-17 (organ growth and maturation). In the 4 and a half-day-old embryo, ODC activity and polyamine concentrations were about twice as high in the head region as compared to the trunk region. In the 14-day-old embryo, the highest ODC and SAMDC activities were found in lung, intestine and kidney, and there was a positive correlation between the enzyme activities and the growth rates of most organs/tissues.     

Polyamine Requirements of a Conditional Polyamine Auxotroph of Escherichia coli

Escherichia coli MA-159 is deficient in agmatine ureohydrolase. After addition of exogenous arginine, the cellular putrescine content declines immediately and exponentially; however, the spermidine content remains normal for 3 h. The growth rate of such cultures, measured turbidometrically, slows gradually over many hours. Putrescine-depleted cultures grow especially slowly in media of low osmolarity, whereas nondepleted cultures grow at similar and rapid rates in media of either normal or low osmolarity. External osmolarity also affects the ability of various exogenous polyamines to stimulate growth of putrescine-depleted cultures. In medium of normal osmolarity, putrescine and spermidine both allow sustained rapid growth for many hours. In low osmolarity medium, putrescine allows sustained rapid growth, whereas cultures containing spermidine grow more slowly; this result cannot be explained by conversion of putrescine to spermidine, for cultures grown with exogenous putrescine contain smaller spermidine pools than do cultures grown with exogenous spermidine. Spermine greatly stimulates growth in medium of normal osmolarity; however, in medium of low osmolarity, spermine is much less effective and can block the action of putrescine. Several other polyamines have been studied in this system. These results confirm and expand previous reports that polyamines are necessary for growth of E. coli and suggest that putrescine may have a specific function during growth in media of low osmolarity.     

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.     

Changes in endogenous polyamine levels are associated with differentiation in Dictyostelium discoideum.

This is the first report correlating levels of polyamines and its fractions with differentiation in Dictyostelium discoideum. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of morphogenesis in this organism. No spermine was found at any given stage and putrescine was the most abundant polyamine. There was a sharp increase in the levels of both free (and total) and conjugated forms of putrescine and spermidine at the slug stage as compared to the growth phase. The levels of putrescine and spermidine were found to be higher in isolated prespore cells as compared to the prestalk cells. Remarkably, the levels of polyamine decreased at the early culminant stage. Data suggest that a moderate level of polyamines is needed for growth but it is important to have high levels of polyamines at the time of differentiation.     

Studies on the accumulation of putrescine and spermidine in Escherichia coli

The rate of accumulation of the polyamines spermidine and putrescine by E. coli depended on growth rate. Spermidine accumulation was faster in chemostat cultures with high dilution rates than in those with low dilution rates and was slower in bacteria that had been grown for several generations with either putrescine or spermidine, suggesting that the spermidine-uptake system was repressed by exogenous polyamines. The uptake of spermidine required metabolic energy. Thus accumulation occurred in an energy-starved unc strain only upon addition of glucose (or D-lactate to a smaller extent). With glucose present accumulation occurred in an unc, frd strain under anaerobic conditions, suggesting that ATP drives uptake. However, accumulation was generally sensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that the proton motive force was involved in uptake. Unlike spermidine, putrescine accumulation was faster in slow-growing than in fast-growing cultures. This may have been due to greater efflux of putrescine at faster growth rates. Accumulation of putrescine was faster following prolonged growth with either putrescine or spermidine, suggesting induction of the putrescine-uptake system by exogenous polyamines. Like spermidine accumulation, putrescine accumulation required metabolic energy. Accumulation was insensitive to CCCP and occurred only when glucose was added to energy-starved unc bacteria, suggesting that high-energy bonds may drive the uptake of putrescine.     

Effects of spermidine synthase inhibition in cultured chick embryo fibroblasts.

The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were also inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis.     

Polyamine Levels in Relation to Growth and Somatic Embryogenesis in Tissue 6Medicago Sativa L.

Polyamine levels in petiole-derived tissue cultures of two highlyregenerable Medicago sativa L. genotypes were determined duringthe embryo induction and embryo differentiation phases of somaticembryogenesis. Putrescine levels increased 27–32-foldduring the 10 d on 2, 4-D- and kinetin-containing embryo inductionmedium and fell sharply following transfer to growth substance-freeembryo differentiation medium. The rapid increase in putrescinecontent occurred during a period of relatively little growthwhile the decline coincided with the initiation of rapid tissuegrowth on embryo differentiation medium. The addition of putativeinhibitors of putrescine or spermidine biosynthesis to the embryoinduction medium led to reduced levels of polyamines, particularlyof putrescine, in cultures of both genotypes but subsequentsomatic embryo formation was inhibited in cultures of one genotypeonly. It was concluded that the pronounced change in putrescinemetabolism was not specifically associated with embryogenesis,but appeared to be related generally to re-programming of cellsinto a new pattern of in vitro development. Key words: Medicago sativa, polyamines, somatic embryogenesis     

Effect of 5''-deoxy-5''-isobutylthioadenosine on putrescine uptake and polyamine biosynthesis by chick embryo fibroblasts.

The effect of 5'-deoxy-5'-S-isobutylthioadenosine (SIBA) on polyamine biosynthesis has been studied by using cultured chick embryo fibroblasts. It has been shown that the drug inhibits the uptake of [14C]putrescine and its conversion into labelled spermidine or spermine. The inhibitory effect is reversed by removing the inhibitor after exposing the cells to the drug for 24 h. SIBA also caused a significant decrease in cellular spermine levels and an accumulation of putrescine. These changes are reversed by removing the inhibitor. SIBA had the same effect on chick embryo fibroblasts transformed by Rous sarcoma virus; a decrease in cellular spermine levels in SIBA-treated cells was observed. In all the experiments SIBA caused a reduction in the spermine/putrescine and spermidine/putrescine ratios. It is suggested that SIBA is not only an inhibitor of transmethylation but also interferes with polyamine biosynthesis, probably by blocking aminopropyltransferase.     

Dystroglycan binding to laminin α1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.     

Effect of inhibitors of polyamine biosynthesis and of exogenous polyamines on the mitogenic effect of epidermal growth factor in a hepatocyte culture

It was established that difluoromethylornithine (DFMO) and methylglyoxal-bis(guanylhydrazone) blocked the mitogenic response of hepatocytes to epidermal growth factor. This effect of inhibitors, especially DFMO, has been prevented by exogenic putrescine, spermidine or spermine. But neither blocking action of inhibitors nor preventing effect of polyamines associated with changes of epidermal growth factor binding to hepatocytes.     

Signaling to the epithelium is not sufficient to mediate all of the effects of transforming growth factor beta and bone morphogenetic protein 4 on murine embryonic lung development.

Many studies have suggested that transforming growth factor beta (TGF-beta) and bone morphogenetic protein 4 (Bmp4) regulate early development of the lung. In this study, administration of growth factors directly into the lumen of lungs grown in organ culture was used to limit their activity to the epithelium and test the hypothesis that signaling to the epithelium is sufficient to mediate the known effects of TGF-beta and BMP-4 on early lung development. Addition of TGF-beta1, beta2, or beta3 to the medium surrounding lungs grown in organ culture resulted in decreased branching, reduced cell proliferation, accumulation of alpha-smooth muscle actin protein (alpha-SMA) in the mesenchyme, and decreased expression of a marker for respiratory epithelium, surfactant protein-C (Sp-C). When TGF-beta1 was restricted to the epithelium, accumulation of alpha-SMA and inhibition of Sp-C expression were not observed but branching and proliferation were inhibited. In contrast, branching was not inhibited in lungs where TGF-beta2 or TGF-beta3 were restricted to the epithelium suggesting differences in the mechanism of signaling by TGF-beta1, TGF-beta2 or TGF -beta3 in lung. Addition of Bmp4 to the medium surrounding lungs grown in organ culture stimulated cell proliferation and branching morphogenesis; however, direct injection of Bmp4 into the lung lumen had no effect on proliferation or branching. Based on these data and data from mesenchyme-free cultures, we propose that the mesenchyme influences growth factor signaling in the lung.