Joseph J. Baldassare

Ras-stimulated Extracellular Signal-related Kinase 1 and RhoA Activities Coordinate Platelet-derived Growth Factor-induced G1 Progression through the Independent Regulation of Cyclin D1 and p27KIP1

Platelet-derived growth factor (PDGF)-induced Ras activation is required for G1 progression in Chinese hamster embryo fibroblasts (IIC9 cells). Ras stimulates both extracellular signal-related kinase (ERK) activation and RhoA activation in response to PDGF stimulation. Inhibition of either of these Ras-stimulated pathways results in growth arrest. We have shown previously that Ras-stimulated ERK activation is essential for the induction and continued G1 expression of cyclin D1. In this study we examine the role of Ras-induced RhoA activity in G1 progression. Unstimulated IIC9 cells expressed high levels of the G1 cyclin-dependent kinase inhibitor p27KIP1. Stimulation with PDGF resulted in a dramatic decrease in p27KIP1 protein expression. This decrease was attributed to increased p27KIP1 protein degradation. Overexpression of dominant-negative forms of Ras or RhoA completely blocked PDGF-induced p27KIP1 degradation, but only dominant-negative Ras inhibited cyclin D1 protein expression. C3 transferase also inhibited PDGF-induced p27KIP1degradation, thus further implicating RhoA in p27KIP1regulation. Overexpression of dominant-negative ERK resulted in inhibition of PDGF-induced cyclin D1 expression but had no effect on PDGF-induced p27KIP1 degradation. These data suggest that Ras coordinates the independent regulation of cyclin D1 and p27KIP1 expression by the respective activation of ERK and RhoA and that these pathways converge to determine the activation state of complexes of cyclin D1 and cyclin-dependent kinase in response to mitogen.

The nuclear transcription factor NF-κB mediates interleukin-1β–induced expression of cyclooxygenase-2 in human myometrial cells

Abstract Objective: Up-regulation of prostaglandin production by gestational tissues in the setting of intrauterine infection has been implicated as an important contributor to preterm labor and parturition. In this study we investigated the possible role of the nuclear transcription factor NF-κB in interleukin-1 signaling, leading to the expression of cyclooxygenase 2 and prostaglandin production in human myometrial cell cultures. Study Design: Human myometrial smooth muscle cells from an immortalized line were used as a model system between passages 20 and 35. Growth-arrested cell cultures were stimulated with human recombinant interleukin 1, and the activation of NF-κB was assessed by the degradation of the inhibitory protein IκB-α (Western analysis), as well as by nuclear binding of NF-κB by using an electrophoretic mobility shift assay. The abundance of cyclooxygenase-2 messenger ribonucleic acid and protein was measured by Northern and Western analyses, whereas prostaglandin (prostaglandin I 2 and prostaglandin E 2 ) production was determined by specific radioimmunoassays. Results: Within 15 minutes of stimulation with interleukin 1, 90% of IκB-α was degraded. This was temporally associated with nuclear translocation and binding of NF-κB. Within 30 minutes, cyclooxygenase 2 messenger ribonucleic acid appeared, with steady-state levels increasing up to 4 hours. This was followed by an up to 80-fold increase in cyclooxygenase 2 protein and a corresponding time-dependent increase in prostaglandin production. When IκB-α degradation was blocked with calpain I inhibitor, NF-κB translocation, cyclooxygenase 2 messenger ribonucleic acid and protein expression, and prostaglandin synthesis were also inhibited. Conclusion: Stimulation of human myometrial cells with interleukin 1 leads to rapid activation of the transcription factor NF-κB, which is functionally linked to the expression of cyclooxygenase 2 messenger ribonucleic acid, protein, and prostaglandin synthesis. (Am J Obstet Gynecol 1999;181:359-66.)

RhoA stimulates p27(Kip) degradation through its regulation of cyclin E/CDK2 activity.

RhoA has been identified as an important regulator of cell proliferation. We recently showed that the Ras/RhoA pathway regulates the degradation of p27Kip and the progression of Chinese hamster embryo fibroblasts (IIC9 cells) through G1 into S phase (Weber, J. D., Hu, W., Jefcoat, S. C., Raben, D. M., and Baldassare, J. J. (1997) J. Biol. Chem.272, 32966–32971). In this report, we have demonstrated that, in IIC9 cells, RhoA regulates cyclin E/CDK2 activity, which is required for p27Kip degradation. As previously shown in several fibroblasts cell lines, expression of dominant-negative CDK2 in IIC9 cells blocked serum-induced cyclin E/CDK2 activity and p27Kip degradation. In the absence of serum, expression of constitutively active RhoA(63) resulted in significant stimulation of cyclin E/CDK2 activity and degradation of p27Kip. Cotransfection of dominant-negative CDK2 and RhoA(63) inhibited RhoA(63)-induced cyclin E/CDK2 activity and p27Kipdegradation. In addition, expression of dominant-negative RhoA blocked serum-induced cyclin E/CDK2 activity and p27Kipdegradation. Finally, expression of catalytically active cyclin E/CDK2 rescued the effect of expression of dominant-negative RhoA. Taken together, these data show that RhoA regulates p27Kipdegradation through its regulation of cyclin E/CDK2 activity.

Nuclear translocation of rhoa mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction

In this paper we demonstrate for the first time a mitogen-induced activation of a nuclear acting phosphatidylcholine-phospholipase D (PLD) which is mediated, at least in part, by the translocation of RhoA to the nucleus. Addition of α-thrombin to quiescent IIC9 cells results in an increase in PLD activity in IIC9 nuclei. This is indicated by an increase in the α-thrombin-induced production of nuclear phosphatidylethanol in quiescent cells incubated in the presence of ethanol as well as an increase in PLD activity in isolated nuclei. Consistent with our previous report (Wright, T. M., Willenberger, S., and Raben, D. M. (1992) Biochem. J. 285, 395-400), the presence of ethanol decreases the α-thrombin-induced production of phosphatidic acid without affecting the induced increase in nuclear diglyceride, indicating that the increase in nuclear PLD activity is responsible for the effect on phosphatidic acid, but not that on diglyceride. Our data further demonstrate that RhoA mediates the activation of nuclear PLD. RhoA translocates to the nucleus in response to α-thrombin. Additionally, PLD activity in nuclei isolated from α-thrombin-treated cells is reduced in a concentration-dependent fashion by incubation with RhoGDI and restored by the addition of prenylated RhoA in the presence of guanosine 5′-3-O-(thio)triphosphate. Western blot analysis indicates that this RhoGDI treatment results in the extraction of RhoA from the nuclear envelope. These data support a role for a RhoA-mediated activation of PLD in our recently described hypothesis, which proposes that a signal transduction cascade exists in the nuclear envelope and represents a novel signal transduction cascade that we have termed NEST (uclear nvelope ignal ransduction).

Fibronectin and cytokines increase JNK, ERK, AP-1 activity, and transin gene expression in rat hepatic stellate cells

Cytokines, growth factors, and alterations in the extracellular matrix composition may play a role in maintaining hepatic stellate cells (HSC) in the activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathways that are stimulated by these factors in HSC remain to be fully elucidated. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) family, including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), plays an important role in the cellular response to stress. The aims of this study were to investigate whether fibronectin (FN) or the inflammatory cytokines interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) activate JNK, ERK, and AP-1 activity in HSC and induce the gene expression of the matrix metalloproteinase transin. Treatment of HSC with FN resulted in an up to 4.5-fold increase in ERK activity and a 2.1-fold increase in JNK activity. IL-1α and TNF-α produced up to a fourfold increase in JNK activity and a twofold increase in ERK activity. We then compared the effects of FN, IL-1α, and TNF-α on AP-1 activity and metalloproteinase mRNA induction. All three compounds increased AP-1 binding and promoter activity, and transin mRNA levels were increased 1.8-fold by FN, 2.2-fold by IL-1α, and 2.8-fold by TNF-α. Therefore, FN and inflammatory cytokines increase MAPK activity, stimulate AP-1 activity, and increase transin gene expression in HSC. Signal transduction pathways involving the MAPK family may play an important role in the regulation of matrix metalloproteinase expression by cytokines and FN in HSC.

Cytokine-Initiated Signal Transduction in Human Myometrial Cells

PROBLEM: The objectives of this study were to evaluate interleukin‐1 (IL‐1) binding and some postreceptor actions of this cytokine and tumor necrosis factor (TNF) in human myometrial cells (HMC).

Direct intra-tumoral injection of zinc-acetate halts tumor growth in a xenograft model of prostate cancer

Intracellular levels of zinc have shown a strong inverse correlation to growth and malignancy of prostate cancer. To date, studies of zinc supplementation in prostate cancer have been equivocal and have not accounted for bioavailability of zinc. Therefore, we hypothesized that direct intra-tumoral injection of zinc could impact prostate cancer growth. In this study, we evaluated the cytotoxic properties of the pH neutral salt zinc acetate on the prostate cancer cell lines PC3, DU145 and LNCaP. Zinc acetate killed prostate cancer cell lines in vitro, independent of androgen sensitivity, in a dose-dependent manner in a range between 200 and 600 μM. Cell death occurred rapidly with 50% cell death by six hours and maximal cell death by 18 hours. We next established a xenograft model of prostate cancer and tested an experimental treatment protocol of direct intra-tumoral injection of zinc acetate. We found that zinc treatments halted the growth of the prostate cancer tumors and substantially extended the survival of the animals, whilst causing no detectable cytoxicity to other tissues. Thus, our studies form a solid proof-of-concept that direct intra-tumoral injection of zinc acetate could be a safe and effective treatment strategy for prostate cancer.

Nuclear Diacylglycerol Kinase-θ is Activated in Response to α-Thrombin

Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with α-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95–108). In this report, we show that the addition of α-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-θ and DGK-δ are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-δ (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394–8401), and constitutively active RhoA inhibits DGK-θ (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820–6822) to identify the activity induced by α-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-θ antibody inhibited the α-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-θ is the isoform responsive to α-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that α-thrombin induced the translocation of DGK-θ to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-θ activity and a nuclear localization of DGK-δ.

Molecular aspects of Huntington's disease

Huntingtons disease (HD) is a progressive neurodegenerative disease striking principally medium spiny GABAergic neurons of the caudate nucleus of the basal ganglia. It affects about one in 10,000 individuals and is transmitted in an autosomal dominant fashion. The molecular basis of the disease is expansion of the trinucleotide CAG in the first exon of a gene on chromosome four. The CAG repeats are translated to polyglutamine repeats in the expressed protein, huntingtin. The normal function of huntingtin remains incompletely characterized, but based upon recently defined protein‐protein interactions, it appears to be associated with the cytoskeleton and required for neurogenesis. Huntingtin has been demonstrated to interact with such proteins as HAP1, HIP1, microtubules, GADPH, calmodulin, and an ubiquitin‐conjugating enzyme. Polyglutamine expansion alters many of these interactions and leads to huntingtin aggregation and the formation of neuronal nuclear inclusions, ultimately culminating in cell death. In this review, we discuss the molecular aspects of HD, including the present understanding of huntingtin‐protein interactions, studies with transgenic mice, and postulated mechanisms of huntingtin aggregation. J. Neurosci. Res. 54:301–308, 1998.

Effects of cholecystokinin (CCK) and other secretagogues on isoforms of protein kinase C (PKC) in pancreatic acini

We used rat pancreatic acini and measured the effects of various agents on digestive enzyme secretion, diacylglycerol (DAG) and the cellular distribution of protein kinase C (PKC) enzyme activity as well as isoforms of PKC determined by quantitative immunoblot analysis. TPA, but not CCK-8, caused translocation of PKC enzyme activity from the cytosol fraction to the membrane fraction. Immunoblot analysis detected PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta. PKC-beta, PKC-gamma and PKC-eta were not detected. TPA caused translocation of all isoforms from cytosol to membrane, whereas CCK-8 caused translocation of PKC-delta and PKC-epsilon, carbachol caused translocation of PKC-epsilon, and bombesin and secretin caused no detectable translocation of any isoform. Specific receptor antagonists could prevent, as well as reverse completely, the translocation of PKC isoforms caused by CCK-8 or carbachol. Agonists added in sequence with an interposed addition of a specific receptor antagonist caused cycling of PKC-epsilon between cytosol and membrane fractions. Each receptor-mediated agonist that caused translocation of PKC also increased DAG, and with CCK-8 and carbachol cycling of PKC-epsilon between cytosol and membrane was accompanied by corresponding cyclic changes in cellular DAG. CCK-JMV-180, bombesin and secretin increased DAG but did not cause translocation of any PKC isoform. Translocation of a PKC isoform could be accounted for by whether the increased DAG originated from PIP2 (accompanied by translocation) or from phosphatidylcholine (no accompanying translocation). Thus it appeared that DAG, in pancreatic acini, is functionally compartmentalized depending on the source of the lipid. Studies using CCK-8 and CCK-JMV-180 indicated that occupation of the low affinity state of the CCK receptor by either peptide increased DAG from phosphatidylcholine, whereas occupation of the very low affinity state by CCK-8 increased DAG from PIP2 and caused translocation of PKC-delta and PKC-epsilon. TPA stimulated amylase secretion, indicating that activation of PKC can stimulate enzyme secretion; however, with the various receptor-mediated secretagogues there was no consistent, unequivocal correlation between translocation of an isoform of PKC and accompanying changes in enzyme secretion.