Growth/differentiation factor-5 induces growth arrest and apoptosis in mouse B lineage cells with modulation by Smad

Bone morphogenetic proteins, including growth/differentiation factor-5 (GDF-5), are multifunctional cytokines. Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta superfamily have focused on Smad proteins. However, scant attention has been given to the mechanism by which GDF-5 exerts its negative growth effect on immunological competent cells. In the present study, we demonstrated that GDF-5 induced cell cycle arrest in the G1 phase before the appearance of apoptosis in mouse B cell hybridoma HS-72 cells, while the ectopic expression of Smad6 and Smad7 in HS-72 cells suppressed the GDF-5-induced G1 cell cycle arrest by abolishing the expression of p21(CIP-1/WAF-1) and hypophosphorylation of retinoblastoma protein. Moreover, we found that Smad6 and Smad7 suppressed GDF-5-induced apoptosis in HS-72 cells. These findings indicated that Smad6 and Smad7 exhibit inhibitory effects toward GDF-5-mediated signaling in B lineage cells.     

Attachment of HeLa cells during early G1 phase

Both growth factor directed and integrin dependent signal transduction were shown to take place directly after completion of mitosis. The local activation of these signal transduction cascades was investigated in early G1 cells. Interestingly, various key signal transduction proteins were found in blebs at the cell membrane within 30 min after mitosis. These membrane blebs appeared in round, mitotic-like cells and disappeared rapidly during spreading of the cells in G1 phase. In addition to tyrosine-phosphorylated proteins, the blebs contained also phosphorylated FAK and phosphorylated MAP kinase. The formation of membrane blebs in round, mitotic cells before cell spreading is not specific for mitotic cells, because similar features were observed in trypsinized cells. Just before cell spreading also these cells exhibited membrane blebs containing active signal transduction proteins. Inhibition of signal transduction did not affect membrane bleb formation, suggesting that the membrane blebs were formed independent of signal transduction.     

RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis.     

Bone morphogenetic protein-7 (OP1) and transforming growth factor-beta1 modulate 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts

Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGFbeta) are potent regulators of osteoblast differentiation and proliferation, processes that are crucial in bone remodeling. BMPs and TGFbeta act in concert with other local factors and hormones, among them 1,25(OH)2-vitamin D3 and insulin. Here we show that BMP7 inhibits 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts, whereas TGFbeta1 stimulates it, as assessed by assays for alkaline phosphatase (ALP) induction, matrix mineralization, and morphology changes. BMP7 or TGFbeta1 alone affects the differentiation of human osteoblasts. Similar results were obtained in assays for ALP induction using conditionally immortalized human osteoblasts (hFOB) and primary osteoblasts obtained from trabecular bone of the femoral head after hip replacement surgery. BMP7 stimulation led to a decrease of 1,25(OH)2-vitamin D3-induced binding of nuclear proteins to a vitamin D response element, as shown by electrophoretic mobility shift assay. Our results suggest that 1,25(OH)2-vitamin D3 modulates in opposite ways the effects of BMP7 and TGFbeta1 on osteoblast differentiation.     

Inhibition of S/G2 phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.     

Potentiation of in vitro lipolytic effect of ACTH by its fragment]

The influence of fragment ACTH 11-14 analogues with amino acid sequences H-Lys-Pro-Val-Gly-OH (fragment I) and H-Lys-Pro-Val-Gly-NH2 (fragment II), possessing structural elements, similar for certain groups of peptide hormones and kinins, on lipolytic effect of ACTH in adipose tissue and isolated epydidymal fat cells of rat, was studied. Both fragments have no effect on the lipolysis; they potentiate the ACTH-induced lipolysis 1,5--2,0 fold, but do not alter the maximal effect at concentrations 0,1--1,0 mkg/ml in tissue and fragment I--at concentrations from 0,01 to 0,1 mkg/ml in isolated fat cell system. The role of "common" fragments in hormone-receptor interactions as well as mechanism of their potentiating effect is discussed. It is assumed that the "common" fragment of ACTH--ACTH11-14--is a second, non-specific active site of hormone directly involved in secondary signal formation.     

Cloning and regulation of a mRNA specifically expressed in the preadipose state

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.     

Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint

Cells eventually exit from mitosis during sustained arrest at the spindle checkpoint, without sister chromatid separation and cytokinesis. The resulting tetraploid cells are arrested in the subsequent G1 phase in a p53-dependent manner by the regulatory function of the postmitotic G1 checkpoint. Here we report how the nucleolus plays a critical role in activation of the postmitotic G1 checkpoint. During mitosis, the nucleolus is disrupted and many nucleolar proteins are translocated from the nucleolus into the cytoplasm. Among the nucleolar factors, Myb-binding protein 1a (MYBBP1A) induces the acetylation and accumulation of p53 by enhancing the interaction between p300 and p53 during prolonged mitosis. MYBBP1A-dependent p53 activation is essential for the postmitotic G1 checkpoint. Thus, our results demonstrate a novel nucleolar function that monitors the prolongation of mitosis and converts its signal into activation of the checkpoint machinery.     

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.     

Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro

This study examines the capability of NIH3T3 fibroblasts to express osteoblastic markers following stimulation with a number of hormones and growth factors in vitro. Of the agents tested, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) dose-dependently induced alkaline phosphatase (ALP) activity in NIH3T3 cells, and this effect was enhanced by the addition of dexamethasone (Dex), which when administered alone caused no detectable ALP expression. The combined use of 1,25(OH)(2)D(3) and Dex also stimulated the synthesis of osteocalcin, and osteopontin. Furthermore, cells treated with the both hormones, in the presence of beta-glycerophosphate and l-ascorbic acid, formed mineralized plaques, indicating an osteoblast (OB) phenotype. By contrast, the differentiation induced by 1,25(OH)(2)D(3) or 1,25(OH)(2)D(3) plus Dex was significantly antagonized by transforming growth factor-beta1 and all trans-retinoic acid. These data indicate that NIH3T3 cells have the potential to adopt an OB-like phenotype and may prove to be a convenient model for studying the early events of osteogenic differentiation and the specific interactions of 1,25(OH)(2)D(3) with glucocorticoids in controlling this process in vitro.     

Effect of head activator on proliferation, head-specific determination and differentiation of epithelial cells in hydra

In hydra the differentiation of head-specific ectodermal epithelial cells from multipotent stem cells is a multistep process in which cell cycle progression is regulated at three restriction points. Head activator acts as a positive signal at these restriction points. At the G2/mitosis boundary of epithelial stem cells head activator functions as a mitogen, being necessary for cell division. Subsequently, in or before S phase, head activator acts as determinant to ensure commitment of epithelial cells to head-specific determination. This effect of head activator requires hundredfold-higher concentrations, and may also require longer incubation times, than for cell proliferation. Epithelial cells thus committed to head-specific differentiation become arrested in G2 as a third and last restriction point in the cell cycle. They require disinhibition by decapitation and probably the presence of head activator for final differentiation, which then occurs in G2.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

Checkpoint kinase 1 prevents cell cycle exit linked to terminal cell differentiation

Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor 4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the cyclin-dependent kinase (CDK)-specific inhibitors p21 and p57, of which p57 is essential for switching from mitotic cell cycles to endocycles. Here, we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a "G2 restriction point" that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.     

Hormone gradients and cartilage cell kinetics

We present a model of growth control in mammalian cartilage growth plates by hormones. The model is based on the distribution of insulin-like growth factors I and II (IGF-I and IGF-II) and their receptors, and assumes that a hormone-receptor complex of IGF controls cells proliferation. A system of differential equations is derived and solved with simplifications in extreme cases, for the one-dimensional time independent case. Even if opposite extremes, such as proliferation control by factors extrinsic to the cell versus intrinsic to the cell, are assumed, similar distributions of hormones and proliferating cells are produced. Hence, choice between alternative models of growth control must be based on empirical observations. On the positive side, similarities between our model for cartilage growth and other models for differentiation and proliferation are evident and might be exploited for unifying these systems on an abstract level.     

Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).     

Epidermal growth factor stimulates DNA synthesis while inhibiting cell multiplication of A-431 carcinoma cells

Epidermal growth factor (EGF) has been shown to inhibit the multiplication of the human epidermoid carcinoma cell line A-431. In the present report it is shown that, despite growth inhibition, EGF caused a marked synthesis of DNA and nonhistone proteins, without progression into mitosis. This event was associated with a retraction of the monolayer into colonies of cells. This suggests that the cell cycle of A-431 cells is controlled by two surface membrane signals: one generated by EGF stimulating the synthetic events of the G1 and S phases; a second signal, leading to progression into mitosis appears either not to be generated or to be inhibited by EGF.     

Cell cycle analysis of sodium butyrate and hydroxyurea, inducers of ectopic hormone production in HeLa cells

Sodium butyrate and hydroxyurea, effective inhibitors of DNA synthesis in HeLa cells, cause these cells to produce increased levels of the ectopic glycopeptide hormones human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), and free alpha chains for these hormones. The objective of this study was an assessment of the role of modulation of cell cycle events in the action of these two chemical agents. A variety of experimental approaches was employed to obtain a clear view of the drugs' effects on cells located initially in all phases of the cell cycle. Cells in early G1, G2, or M phase at time of addition of either inhibitor were not arrested at early time points, but by 48 hours became collected at a location characteristic for each drug, near the G1-S phase boundary. Flow microfluorometry (FMF) and thymidine labeling index revealed that butyrate-treated cells arrested late in G1 phase very close to S phase, while hydroxyurea-blocked cells continued to early S phase. Both inhibitors prevented cells originally in S phase from reaching mitosis. S cells exposed to hydroxyurea were killed by 48 hours, but those growing in 5 mM butyrate progressed to the end of S or G2 phase where they became irreversibly arrested although not removed from the monolayer. Analysis of the cell cycle location and viability of each subpopulation resulting from 48 hour exposure to butyrate or hydroxyurea is important for the study of the function of each cellular subset. Treatment of HeLa cells with lower concentrations of butyrate (1 mM) resulted in slowed yet exponential growth. Fraction labeled mitosis (FLM) analysis shows that this is a result of prolongation of the G1 phase.