Pregnancy-associated plasma protein-A regulates myoblast proliferation and differentiation through an insulin-like growth factor-dependent mechanism

Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAPP-A increased their proliferation in a dose- and time-dependent manner. Addition of exogenous PAPP-A also increased the myotube formation and the activity of creatine kinase in C2C12 cultures. Transient overexpression of the full-length PAPP-A-(1-1547), but not truncated protease-inactive N-terminal PAPP-A-(1-920) or C-terminal PAPP-A-(1100-1547), significantly enhanced the proliferation of C2C12 myoblasts. In vitro and in situ experiments demonstrated that PAPP-A cleaves insulin-like growth factor-binding protein (IGFBP)-2, but not IGFBP-3, in the conditioned medium of C2C12 myoblasts. Overexpression of PAPP-A led to degradation of the IGFBP-2 produced by C2C12 myoblasts and increased free IGF-I concentrations without affecting total IGF-I concentrations. Addition of protease-resistant IGFBP-4 completely abolished the PAPP-A-induced proliferation of C2C12 myoblasts. Our results demonstrate that 1) PAPP-A increases the proliferation and differentiation of myoblasts, 2) the stimulatory effect of PAPP-A on myogenesis is governed by its proteolytic activity, and 3) PAPP-A promotes skeletal myogenesis by increasing the amount of free IGFs via specific degradation of IGFBP-2 produced by myoblasts.     

Inhibition of the proteolytic activity of pregnancy-associated plasma protein-A by targeting substrate exosite binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.     

Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro.

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.     

Cellular mechanism for norepinephrine suppression of pineal photoreceptor-like cell differentiation in rat pineal cultures.

Although the rat pineal is an endocrine organ and has no photoreceptor activity, pineals from neonatal rats contain cells that can differentiate into rod-like cells with rhodopsin immunoreactivity (Rho-I), when cultured in vitro. Norepinephrine (NE) reduces the number of Rho-I cells in a dose-dependent manner and has a considerable effect even at 20 nM. When cultured in vitro, pineals removed up to Postnatal Day 4 differentiated into Rho-I cells to the same extent as did those removed at Day 1 (neonatal), but those removed at Day 5 showed a sharp reduction in the number of differentiated Rho-I cells. This suggests that either pineal cells in situ lose their potential to differentiate by Day 5 or the subpopulation of cells involved normally disappears in pineals older than Day 5. The effect of NE was examined in cultures of neonatal pineals by administering it for 1 or 2 days at different stages during a 9-day culture period. NE was most effective when present in the culture medium at an early culture phase and was not efficacious if present only later than Culture Day 7. This indicates that presumptive pineal photoreceptors may become sensitive to NE only for a limited period and that once they are exposed to NE within this period they are irreversibly affected, possibly to degenerate. These cells are similarly and severely affected by potassium ion concentrations as low as 15 mM, suggesting that NE may act at the adrenoreceptor to modify the membrane properties. Serotonin-immunoreactive cells, another cell type (endocrine) found in the cultures, appeared to be regulated by NE by a separate mechanism. NE suppresses process extension by serotonin cells in a reversible manner, and KCl does not have this effect. These findings further evidence that neurotransmitters may have essential roles, other than the transmission of signals, in modulating the developing nervous system.     

Fluorescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations

Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone-related protein receptor (PTH1R) as osteoblast markers in dual-color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP(-)PTH1R(-), ALP(+)PTH1R(-), ALP(-)PTH1R(+), and ALP(+)PTH1R(+) populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities ( approximately 150-8,500 cell/cm(2); n = 3). Our results indicate that colony forming unit-osteoblast (CFU-O)/bone nodule-forming cells are found in all fractions, but the frequency of CFU-O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP(-)PTH1R(-), ALP(-)PTH1R(+), ALP(+)PTH1R(-), and ALP(+)PTH1R(+) cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU-O number in the four fractions, with the largest stimulation in the ALP(-) cell populations. However, there was no significant difference in the number or size distribution of CFU-F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non-autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP(-)PTH1R(-) population was capable of forming nodules at the clonal level, at approximately 3- or 12-times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity.     

Control of cell proliferation and differentiation by regulating polyamine biosynthesis in cultures of Brassica and its correlation with glyoxalase-I activity

Differentiation in Brussica cultures could be induced on basal medium lacking hormones, while addition of hormones (NAA, BA) resulted in profuse callusing without any differentiation. Supplementing the hormone medium with spermidine resulted in increase in the fresh weight and glyoxalase-I activity by 330% and 8-fold, respectively. Omission of hormones caused spermidine to be less effective in inducing either cell proliferation or differentiation. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, had a retarding effect on callus induction and division of cells in suspension cultures but lead to differentiation and inhibited glyoxalase-I activity. The ability of spermidine to overcome MGBG enhanced differentiation was probably through the breaking of cell cycle arrest. Addition of glutathione, a coenzyme for glyoxalase-I enzyme, promoted cell division and enzyme activity both in callus and suspension cultures. pH emerged as an important factor in controlling glyoxalase-I activity and cell division. Results indicate involvement of spermidine in cell proliferation and differentiation and its correlation with glyoxalase-I activity.     

肿瘤细胞恶性增殖和细胞周期调控改变的分子机制

真核细胞通过复杂的细胞周期调控系统控制细胞的分裂,从而维持有机体的正常代谢和增殖.细胞周期的调控是由一系列重要的信号分子和周期蛋白家族来完成的,这些调节因子发生突变或者表达水平发生改变,将导致细胞周期调控的改变,使细胞增殖能力增强、分化减弱,丧失细胞原有的功能,最终发展成肿瘤细胞.因此细胞周期及其相关调控蛋白和信号机制成为抗肿瘤研究的热点.     

Defined protein-free NS0 myeloma cell cultures: stimulation of proliferation by conditioned medium factors

A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, beta-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 x 10(6) cells mL(-1) in this medium. The antibody yield was 195 mg L(-1) in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 x 10(6) cells mL(-1), had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.     

Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor β (TGF-β) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-β binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-β binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-β activity, on TGF-β binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting. J. Cell. Biochem. 67:528–540, 1997. © 1997 Wiley-Liss, Inc.     

Immunophenotype and sister chromatid differentiation: a combined methodology for analyzing cell proliferation in unfractionated lymphocyte cultures

Combination of the MAC (morphology, antibody, chromosomes) and harlequin staining procedures offers a method for direct analysis of cell kinetics in cultures of unfractionated hematopoietic cells. In the present study unfractionated human mononuclear leukocyte cultures were stimulated with PHA or PWM mitogens and exposed to bromodeoxyuridine for various periods. For MAC, cytospin preparations were made and cells were classified with monoclonal B and T antibodies by the immunoperoxidase technique. After differentiation of the different lymphocyte subsets, the cells were stained by a fluorescence-plus-Giemsa method to distinguish sister chromatids and to determine the proportions of first, second, third, or subsequent mitoses among the previously identified subsets. The results showed (1) that the relative proportions of mitotic T and B cells are the same regardless of the mitogen used; (2) that T and B lymphocytes proliferate faster in cultures stimulated by PWM than in those stimulated by PHA; and (3) that T cells enter mitosis earlier than B cells when PHA or PWM are used as mitogens.     

Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.     

Cell proliferation and cell differentiation in tissue cultures of adult muco-cilliary epithelia.

Explants and monolayers from a variety of muco-ciliary epithelia were cultivated in vitro and the kinetics of their proliferation and differentiation described. New epithelial lining and epithelial-like monolayer sheets of cells formed in which the migration cells were all originally undifferentiated cycling stem cells. The divided and differentiated in ML growth into cell types characteristic of the tissue source: however, the control mechanisms which regulate cell division and cell differentiation in the tissues were lost outside the tissue framework. Cell division and cyto-differentiation in ML growths both in ciliated and in mucus-producing cells, were not always mutually exclusive.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Poly(ADPR)polymerase expression and activity during proliferation and differentiation of rat astrocyte and neuronal cultures.

Poly(ADPR)polymerase (poly(ADPR)P) mRNA and enzymatic activity levels were investigated in primary cultures of rat astrocytes and neurons in the absence or presence of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), respectively. In cultured rat astrocytes, a biphasic increase in poly(ADPR)P mRNA, associated with enhanced nuclear poly(ADPR)P enzymatic activity, were observed. The first rise in poly(ADPR)P mRNA and enzymatic activity is at the beginning of cell proliferation and the second with the occurrence of cell differentiation. In the presence of bFGF (5 ng/ml) the mRNA peaks and the differentiation-associated poly(ADPR)P enzymatic activity undergoes a 2-fold increase. In neuronal cultures an initial high level of poly(ADPR)P mRNA is followed by a decrease while differentiation is progressively achieved. A limited increase of poly(ADPR)P activity is observed during this phase. In the presence of NGF (50 ng/ml), similar poly(ADPR)P mRNA expression and enzymatic activity patterns are observed. The results suggest that poly(ADPR)P is involved at the onset of nerve-cell proliferation and differentiation.     

FoxO调控细胞增殖、分化与凋亡

与细胞发育和代谢相关的转录因子中,2000年才正式发布并统一命名的Fox家族受到了研究者的高度重视,其广泛存在于从酵母到哺乳类的真核生物中.FoxO转录因子作为Fox家族主要成员,是INS/IGF-1信号通路中的关键因子,通过转录调控和信号转导途径在动物的生理调节、代谢和细胞周期等方面起重要作用.     

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.     

Pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the mechanism of IGFBP-4 proteolysis by PAPP-A

The kinin system has been recognized as a locally operating hormone system of cardiovascular cells, however, the molecular mechanisms regulating circumscribed kinin release on cell surfaces are not fully understood. In particular, the principal cell docking sites for the kinin precursor, high molecular weight kininogen (HK), are not fully explored. Here we demonstrate by enzymatic digestion, recombinant overexpression, and affinity cross-linking studies that cell surface chondroitin sulfate (CS) chains of proteoglycans (PGs) serve as major HK binding sites on platelet, fibroblast, liver, and endothelial kidney cells. In this way, CS-type PGs may contribute to a local accumulation of kinin precursors on cell surfaces and modulate circumscribed release of short-lived kinin hormones at or next to their site of action.     

Bioinsecticide activity, bacterial cell lysis and proteolytic activity in cultures of Bacillus thuringiensis subsp. israelensis

Pattern of bioinsecticide activity and cell lysis were monitored in a range of media. Maximum cell lysis, usually exceeding 90% of total cells, occurred at or after the point of maximum bioactivity. In the majority of media tested, insecticide synthesis and cell lysis phases occurred between 24 and 48 h, although they were between 12 and 24 h in some media and later in others. In some media a decline in insecticidal activity was observed after maximum bioactivity was reached. Bioactivity produced per 10⁹ spores ranged from 0.10 to 0.23 mg of purified insecticidal crystal and 3.4 to 5.8 times 10⁴ international toxic units. When proteolytic activity was monitored in cell cultures, extracellular endoprotease was observed before cell lysis and, in addition, there was a linear rise in activity during cell lysis. Aminopeptidase activity appeared in cell free supernatant fluids only during the cell lysis stage. Optima pH for endoprotease and aminopeptidase were 8.5 and 10.25 respectively. The decline in bioinsecticide activity observed may be due to proteolytic action.