Fresh Serum-containing Medium Research Articles

Radial Spoke Protein 3 Is a Mammalian Protein Kinase A-anchoring Protein That Binds ERK1/2

Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIalpha and RIIbeta, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes.

Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

Akt Stimulates the Transactivation Potential of the RelA/p65 Subunit of NF-κB through Utilization of the IκB Kinase and Activation of the Mitogen-activated Protein Kinase p38

The serine/threonine kinase Akt/PKB is a potent regulator of cell survival and has oncogenic transformation potential. Previously, it has been shown that Akt can activate the transcription factor NF-kappaB and that this functions to block apoptosis induced by certain stimuli. The mechanism whereby Akt activates NF-kappaB has been controversial, with evidence supporting induction of nuclear translocation of NF-kappaB via activation of IkappaB kinase activity and/or the stimulation of the transcription function of NF-kappaB. Here we demonstrate that Akt targets the transactivation function of NF-kappaB by stimulating the transactivation domain of RelA/p65 in a manner that is dependent on IkappaB kinase beta activity and on the mitogen-activated protein kinase p38 (p38). Activation of RelA/p65 transactivation function requires serines 529 and 536, sites shown previously to be inducibly phosphorylated. Consistent with the requirement of p38 in the activation of NF-kappaB transcriptional function, expression of activated Akt induces p38 activity. Furthermore, the ability of IL-1beta to activate NF-kappaB is known to involve Akt, and we show here that IL-1beta induces p38 activity in manner dependent on Akt and IkappaB kinase activation. Interestingly, activated Akt and the transcriptional co-activators CBP/p300 synergize in the activation of the RelA/p65 transactivation domain, and this synergy is blocked by p38 inhibitors. These studies demonstrate that Akt, functioning through IkappaB kinase and p38, induces the transcription function of NF-kappaB by stimulating the RelA/p65 transactivation subunit of NF-kappaB.

Characterization of novel phorbol ester- and serum-responsive sequences of the rat ornithine decarboxylase gene promoter.

Ornithine decarboxylase (ODC), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promoters. We used transient expression assays and DNA-protein binding studies to examine the regulation of ODC promoter activity by phorbol esters and serum growth factors. A fragment of the ODC 5' flanking region (nt-1156 to +13) was sufficient to confer 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive expression to a luciferase reporter gene when transfected into H35 cells. However, induction by TPA was not observed in Rat2 fibroblasts, although refeeding of serum-starved Rat2 cells with fresh serum-containing medium rapidly induced a fivefold to sixfold increase in ODC promoter activity, maximal about 8 h after refeeding. Deletion analysis demonstrated that several sequences contributed to basal ODC promoter activity but that nt -92 to +13 was sufficient for induction by TPA or by serum. This sequence lacked canonical TPA-responsive elements, and an activator protein-1 (AP-1) consensus oligonucleotide failed to compete effectively for proteins binding to this region. Two of four protein complexes observed by gel-shift analysis of nt -92 to +13 were competitively inhibited by wild-type but not mutant oligonucleotides encompassing a variant cyclic AMP-response element (CRE) (ODC nt -50 to -42); however, a consensus CRE did not compete. Mutagenesis of this site demonstrated that it contributes to basal expression of the ODC promoter but not to TPA or serum responsiveness. Thus, we conclude that the proximal ODC promoter (nt -92 to +13) responds to TPA and serum stimulation in a cell-type-specific manner that is not mediated by canonical AP-1 elements.

Involvement of cell cycle and mitogen-activated pathways in induction of parathyroid hormone-related protein gene expression in rat aortic smooth muscle cells.

PTH-related protein (PTHrP) is induced in aortic vascular smooth muscle cells (VSMC) in association with mitogen-stimulated proliferation. In this study we examined the role of the cell cycle in the control of PTHrP gene expression. In asynchronously cycling cells grown in serum-containing medium, PTHrP-immunoreactive cells were enriched in G2+M, as revealed by fluorescence-activated cell sorting using a specific monoclonal antibody. PTHrP messenger RNA (mRNA) increased transiently in cells after release from chemically induced cell cycle blockade; levels increased by 10-fold at 2 h, coincident with expression of histone-4 mRNA and enrichment of VSMC in the early S phase. However, PTHrP mRNA levels then declined abruptly while the proportion of cells in the S phase and histone-4 mRNA levels remained constant for 8 h. When cell cycle-arrested cells were exposed to fresh serum-containing medium, angiotensin-II, or phorbol ester without removing the cell cycle blocking agents, PTHrP mRNA levels were induced over a time course identical to that observed in cells released from the blockade. This suggests that progression through the cycle per se is not necessary for mitogen-induced PTHrP mRNA expression, and that conventional chemical synchronization is not adequate to examine the cell cycle dependency of PTHrP mRNA abundance in VSMC. By contrast, in two different PTHrP-producing carcinoma cell lines, PTHrP and its mRNA were not altered as a function of cell cycle, demonstrating that different mechanisms control PTHrP expression in these cancer cells. In conclusion, constitutive immunoreactive levels of PTHrP are low in normally cycling VSMC (but not cancer cells) and accumulate during the latter stages of the cell cycle, suggesting a role for this protein in the process of smooth muscle cell division. However, separate mechanisms, which are independent of cell cycle, operate through a protein kinase-C-dependent pathway(s) to mediate the stimulation of PTHrP gene expression by vasoconstrictors such as angiotensin-II.

Growth of Chinese hamster ovary (CHO) cells expressing the 5-HT 2 serotonin receptor in suspension culture: an efficient method for large-scale acquisition of membrane protein for drug evaluation

Chinese hamster ovary (CHO) cells expressing the rat 5-HT 2 serotonin receptor were grown and evaluated in suspension culture to provide an efficient method of producing membrane-bound receptors for drug discovery. Expression of the 5-HT 2 receptor in cells grown in batch suspension culture fluctuated as a function of culture age. Both receptor expression and receptor G-protein coupling were the highest early (1.9 pmol/mg membrane protein) but declined rapidly as the culture increased in age. However, addition of fresh serum-containing medium to stationary-phase cells reversed the decline and, after 24 h of growth, resulted in maximal receptor density and G-protein coupling. This serum response was found to be reproducible and lead to the establishment of a semi-continuous batch culture system in which cells were maintained in a growth state that supported high levels of membrane-incorporated and G protein-coupled receptors. In this system, 50% of the culture volume could be removed at 24-h intervals for membrane preparation and the lost volume replenished with fresh medium, resulting in a continuous supply of high-quality membrane preparations.

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K + or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.

Alterations in the phosphorylation and activity of DNA polymerase α correlate with the change in replicative DNA synthesis as quiescent cells re-enter the cell cycle

The regulation of DNA polymerase alpha was examined in quiescent, human fibroblast cells stimulated to re-enter the cell cycle by subculturing in fresh serum-containing medium. The level of DNA polymerase alpha activity was measured in cell lysates and after specific immunoprecipitation. DNA polymerase alpha activity increased approximately 10-fold during the period of measurement. The activity increase was coincident with an approximately 60-fold increase in thymidine incorporation in the whole cells representing the first S phase. The large increase in polymerase alpha activity was not predominantly the result of synthesis of new polymerase, since the abundance of the enzyme changed less than 2-fold over the measured period. The quantity of [32P]phosphate incorporated into two subunits (180 and 68 kilodaltons) of DNA polymerase alpha increased approximately 10-fold in parallel with the increase in polymerase activity. The specific activity of the cellular ATP pool remained nearly constant over the period of measurement, indicating that the increase in labeling reflects a true increase in incorporation of phosphate. Results from other laboratories indicate that phosphorylation of DNA polymerase alpha increases its catalytic activity. Our results then suggest that the activity increase observed in DNA polymerase alpha when quiescent, human fibroblasts are stimulated to proliferate is largely caused by a phosphorylation-dependent regulatory process.

Characterization and differential expression of an endothelial cell-specific surface antigen in continuous and sinusoidal endothelial, in skin vascular lesions and in vitro.

Continuous and sinusoidal endothelial cells display marked morphological and functional heterogeneity as to their plasmalemmal vesicle content, to the kind of intercellular junctional complexes, to the existence and kind of fenestrae and gaps, to the existence and character of their basement membrane, to their ability for phagocytosis and to other functional parameters. Monoclonal antibody 1F10, raised against human umbilical vein endothelial cells (HUVE cells), reflects these differences in recognizing--without any nonendothelial side reactions--an endothelial cell surface antigen, abundantly expressed in continuous endothelia, low and inconsistently expressed in liver sinusoidal and dermal lymphatic endothelia and absent from splenic sinusoidal endothelial cells. In differentiated skin vascular tumors, 1F10 antigen is expressed in normal amounts while it is only low and inconsistently expressed in the dedifferentiated endothelial cells of Kaposi's sarcoma and hemangiosarcoma. HUVE cells in culture, in contrast to their in situ ancestors, express variable amounts of 1F10 antigen. When endothelial cell-conditioned medium (ECC medium) is supplied to HUVE cells in culture, no 1F10 antigen is expressed, while supplementation with fresh serum-containing medium (FSC medium) or cytokines, such as bFGF, suffices to maintain 1F10 expression in 10-70% of the cells. From this we conclude that developmental regulation, environmental influences and cytokine supply contribute to the differentiation and maintenance of the 1F10+ and 1F10-endothelial cell phenotypes, both in vivo and in vitro.

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line, RTG-2.

Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G1-S transition to be studied in a representative of teleosts.

Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: characterization and comparison to quiescent cell inhibitor.

Cell membranes prepared from senescent human diploid fibroblasts (HDF) inhibited entry into S phase by 35% when added to the medium of replicating young HDF. This membrane-associated inhibitory activity was (i) sensitive to trypsin, heat, and periodate, which suggests that the inhibitor is a glycoprotein, and (ii) not able to inhibit DNA synthesis in simian virus 40-transformed HDF, which indicates that not all types of cells are sensitive to this inhibitor. Quiescent young HDF also have a surface membrane-associated inhibitor of DNA synthesis. A comparison of the senescent HDF and quiescent HDF inhibitor activities indicates that they may have the same chemical and physical nature and the same specific activity, but their regulation is different. The inhibitory activity of quiescent young HDF is abolished within 20 hr after refeeding with fresh serum-containing medium, whereas that of senescent HDF remains unchanged. Quiescent old HDF (two or three population doublings remaining) exhibit an intermediate response to serum with approximately two-thirds of the inhibitory activity abolished. The fraction of cells in S phase at 20-24 hr post-stimulation (37% in young HDF, 24% in old HDF, and 0% in senescent HDF) is inversely proportional to inhibitor levels. This suggests that inability to neutralize the inhibitory activity in response to serum stimulation could be involved in the inability of senescent HDF to enter S phase. Disappearance of the inhibitory activity from quiescent young HDF occurs late in G1 phase. Thus, the inhibitor may play a role in determining the length of the G0 to S phase transition in these cells.

Regulation of initiation factors during translational repression caused by serum depletion. Covalent modification.

One to 2 h after transfer of HeLa cells into fresh serum-containing medium, when translation rates are maximal, the initiation factor proteins were examined on immunoblots of two-dimensional gels. Eukaryotic initiation factor (eIF)-2 alpha, eIF-2 beta, and eIF-4A each formed a single immunoreactive spot; eIF-2 gamma formed 2 spots; and eIF-4B formed a complex array of 12-20 spots. After 4 days of growth in unreplenished medium, when translation rates have dropped 4-6-fold, several alterations in the isoelectric forms were observed: eIF-2 alpha now occurred in 2 forms, eIF-2 beta was present in 3-4 forms, and the most acidic cluster of eIF-4B variants was decreased or absent while a new isoelectric variant appeared at the basic end of the array. No changes were observed for eIF-2 gamma or eIF-4A. The 35-50-kDa subunits of the multiprotein initiation factor eIF-3 also showed no changes when the aforementioned growth states were compared. Resolution of 32P-labeled lysates by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the eIF-2 alpha modification and the loss of eIF-4B variants reflected changes in phosphorylation states. Stimulation of 4-day grown cells with fresh serum-containing medium caused a reversal of the initiation factor modifications back to the forms prevailing shortly after replating. This analysis indicates that covalent modifications appear concurrently with decreasing initiation rates and suggests that they may be causative.

Effect of soluble products from lectin-stimulated lymphocytes on the growth, adhesiveness, and glycosaminoglycan synthesis of cultured synovial fibroblastic cells.

Human blood mononuclear cells exposed to concanavalin A or phytohemagglutinin secrete a soluble factor that arrests the growth of human synovial fibroblastic cells in culture. Once the growth-inhibitory effect is initiated it cannot be reversed by washing the fibroblastic cells, by refeeding with nonconditioned fresh serum-containing medium, by trypsinization, EDTA treatment, or a combination of these procedures. Media from nonstimulated mononuclear cells, fibroblastic cells, or the lectins themselves do not contain similar inhibitory activity that can be detected by the present culture systems. This lectin-dependent, growth-inhibitory activity does not have a cytotoxic effect on the fibroblasts but increases their adhesiveness to plastic or glass surfaces, and the cells tend to assume a less fibroblastic morphology. The growth-inhibitory activity is stable in the cold and is nondialyzable or ultrafilterable, but the activity is rapidly lost at temperature between 60 degrees and 70 degrees C and at pH 2.0. The growth-arrested cells secrete more glycosaminoglycan per cell in the medium and synthesize more cell surface glycosaminoglycan than the controls. However, the increased glycosaminoglycan synthesis cannot be explained as being entirely secondary to a cell density effect as it is also observed when adjustments are made for the differences in growth rates.

Independent expression of avian sarcoma virus in doubly infected chicken embryo fibroblasts.

Infection of a chicken cell with avian sarcoma virus requires division of the infected cell before synthesis of infectious progeny is initiated. This requirement for a cell division for the complete expression of avian sarcoma virus has been examined further with chicken embryo fibroblasts infected with two distinct viruses. Chicken cells infected with and producing a mutant of Rous sarcoma virus temperature sensitive for transformation (tsLA24PR-A) were arrested in G0 by depletion of serum factors from growth medium. These stationary cells continued to produce infectious progeny in the absence of further cell division. Superinfection of the stationary cells with the wild-type Prague strain of Rous sarcoma virus (PR-RSV-C) produced a stable double infection in these cells. Progeny of the superinfecting PR-RSV-C, however, were not detected until these cells underwent division after stimulation with fresh serum-containing medium. The addition of colchicine to these serum-stimulated cells, although not affecting production of the tsLA24PR-A, inhibited the appearance of progeny of the superinfecting PR-RSV-C. These experiments indicate that each avian sarcoma virus infection of a chicken embryo fibroblast requires division of the infected cell for production of that virus regardless of whether or not the cell is already producing a similar virus. The results suggest, therefore, that the requirement for a cell division represents a requirement for an event that controls virus expression in a "cis-acting" fashion specific for the provirus.

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [ 3H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [ 3H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [ 3H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.

Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA).

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.