Larry R. Karns

Cloning of the rat alpha 1C-adrenergic receptor from cardiac myocytes. alpha 1C, alpha 1B, and alpha 1D mRNAs are present in cardiac myocytes but not in cardiac fibroblasts.

alpha 1-Adrenergic receptor (AR) activation in cardiac muscle has several different physiological effects that might be mediated through different alpha 1-AR subtypes. Two alpha 1-AR subtypes have been cloned from the rat, the alpha 1B and the alpha 1D; both are present in adult rat heart. A third subtype, the alpha 1C, cloned from the cow and human, was reported to be absent in the rat. However, we recently found alpha 1C mRNA in adult rat heart by using a partial alpha 1C cDNA. Thus, all three cloned alpha 1-AR subtypes are present in the heart, but it is unknown whether each is expressed in cardiac myocytes or in cardiac fibroblasts. In the present study, the full-length rat alpha 1C-AR was cloned from cultured neonatal cardiac myocytes. alpha 1C mRNA transcripts of 3, 9.5, and 11 kb were present in adult rat heart by Northern blot analysis. alpha 1B-, alpha 1C-, and alpha 1D-subtype mRNAs were each present in isolated adult and neonatal cardiac myocytes by RNase protection assay. In addition, cultured neonatal cardiac myocytes expressed the three alpha 1-AR subtype mRNAs. In contrast, none of the alpha 1-AR mRNAs was detected in cultured neonatal cardiac fibroblasts. In addition, alpha 1-ARs were absent in fibroblasts by [3H]prazosin binding and norepinephrine-stimulated [3H]inositol phosphate production. The absence of alpha 1-ARs in cardiac fibroblasts differs from beta-adrenergic and angiotensin II receptors, which are present in both cardiac fibroblasts and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-1 content and pattern of expression correlates with histopathologic grade in diffusely infiltrating astrocytomas.

Studies of experimental tumorigenesis have strongly implicated signaling of the insulin-like growth factor 1 (IGF-1) as a key component in astrocytic neoplasia; however, its role in the growth of low-grade and malignant human tumors is not well understood. Correlative analyses of IGF-1, p53, and Ki-67 (MIB-1) immunohistochemistry and IGF-1 receptor (IGF-1R) mRNA expression were performed to examine the cellular pattern of IGF-1 signaling in 39 cases of astrocytoma (World Health Organization grades II-IV). Tumor cells expressing IGF-1 and IGF-1R were present in all tumor grades. The proportion of tumor cells that expressed IGF-1 correlated with both histopathologic grade and Ki-67 labeling indices, while expression of IGF-1R mRNA correlated with Ki-67 indices. In cases where stereotactic tissue sampling could be identified with a specific tumor area by neuroimaging features, the numbers of IGF-1 immunoreactive cells correlated with the tumor zones of highest cellularity and Ki-67 labeling. In glioblastomas, the localization of IGF-1 immunoreactivity was notable for several features: frequent accentuation in the perivascular tumor cells surrounding microvascular hyperplasia; increased levels in reactive astrocytes at the margins of tumor infiltration; and selective expression in microvascular cells exhibiting endothelial/pericytic hyperplasia. IGF-1R expression was particularly prominent in tumor cells adjacent to both microvascular hyperplasia and palisading necrosis. These data suggest that IGF-1 signaling occurs early in astroglial tumorigenesis in the setting of cell proliferation. The distinctive correlative patterns of IGF-1 and IGF-1R expression in glioblastomas also suggest that IGF-1 signaling has an association with the development of malignant phenotypes related to aberrant angiogenesis and invasive tumor interactions with reactive brain.

Phorbol 12-Myristate 13-Acetate Induces Protein Kinase Cη-specific Proliferative Response in Astrocytic Tumor Cells

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-η expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (α, βI, βII, and γ) and novel (θ and ε) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-η, was only expressed by U-251 MG cells. In contrast, PKC-δ was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-η was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-η cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 μm) and the MEK inhibitor, PD 98059 (50 μm). Transient transfection of wild type U-251 with PKC-η antisense oligonucleotide (1 μm) also blocked the PMA-induced increase in [3H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-η plays a determining role in the different proliferative capacity.

Transcriptional regulation of LDL receptor-related protein by IFN-gamma and the antagonistic activity of TGF-beta(1) in the RAW 264.7 macrophage-like cell line.

Low density lipoprotein receptor‐related protein (LRP) is a major receptor for multiple ligands, including chylomicron and VLDL remnants, bacterial toxins, viruses, proteinases, lipoprotein lipase, and activated α2‐macroglobulin (α2M). In this study, we used Northern blot analyses and nuclear run‐on experiments to demonstrate that interferon‐γ (IFN‐γ) causes a concentration‐dependent decrease in steady‐state LRP mRNA expression and gene transcription rate in RAW 264.7 cells. IFN‐γ also markedly increased expression of inducible nitric oxide synthase (NOS), as expected; however, the increase in nitric oxide was not responsible for the down‐regulation of LRP expression since the NOS inhibitor, N G‐monomethyl‐l‐arginine, did not preserve LRP expression in IFN‐γ‐treated cells. Trans‐forming growth factor‐β1 (TGF‐β1; 2.5 ng/mL) had no independent effect on LRP expression and did not modify the response to IFN‐γ when the two cytokines were added simultaneously to cultures. When TGF‐β1 was added 24 h prior to IFN‐γ, the extent of LRP down‐regulation was significantly reduced. Specific binding of the LRP ligand, activated 125I‐α2M, was decreased by 76 ± 5% in cells treated with 100 U/mL IFN‐γ, but only by 45 ± 7% in cells treated with 100 U/mL IFN‐γ after TGF‐β1‐pretreatment. The antagonistic activity of TGF‐β1 on the IFN‐γ response in RAW 264.7 cells did not result from a change in LRP mRNA stability or IFN‐γ receptor expression, as determined by Northern blot analyses 125I‐IFN‐γ binding experiments. The studies presented here suggest that the balance between IFN‐γ and TGF‐β1 may be critical in determining LRP expression at sites of infection and inflammation.

Localization of the Binding Site for Transforming Growth Factor-β in Human α2-Macroglobulin to a 20-kDa Peptide That Also Contains the Bait Region

α2-Macroglobulin (α2M) functions as a major carrier of transforming growth factor-β (TGF-β) in vivo. The goal of this investigation was to characterize the TGF-β-binding site in α2M. Human α2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-β1, TGF-β2, and NGF-β in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-β-binding site in α2M, five cDNA fragments, collectively encoding amino acids 122–1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-β2 bound only to the fusion protein (FP3) that includes amino acids 614–797. FP3 bound125I-TGF-β1 and 125I-TGF-β2 in solution, preventing the binding of these growth factors to immobilized α2M-methylamine (α2M-MA). The IC50 values were 33 ± 5 and 26 ± 6 nm for TGF-β1 and TGF-β2, respectively; these values were comparable with or lower than those determined with native α2M or α2M-MA. A GST fusion protein that includes amino acids 798–1082 of α2M (FP4) and purified GST did not inhibit the binding of TGF-β to immobilized α2M-MA. FP3 (0.2 μm) neutralized the activity of TGF-β1 and TGF-β2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an α2M activity that has been attributed to the neutralization of endogenously synthesized TGF-β. Thus, we have isolated a peptide corresponding to 13% of the α2M sequence that binds TGF-β and neutralizes the activity of TGF-β in two separate biological assays.

Characterization of the Binding Sites for Plasminogen and Tissue-Type Plasminogen Activator in Cytokeratin 8 and Cytokeratin 18

Cytokeratin 8 (CK8) is an intermediate filament protein that penetrates to the external surfaces of breast cancer cells and is released from cells in the form of soluble heteropolymers. CK8 binds plasminogen and tissue-type plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. In this study, we prepared GST-fusion proteins which contained either 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids from the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identical to CK8f except that the C-terminal lysine was mutated to glutamine (CK8fK483Q). CK8f bound plasminogen; the KD was 0.5 μM. Binding was completely inhibited by εACA. CK8fK483Q also bound plasminogen, albeit with decreased affinity (KD ≍ 1.5 μM). CK18f did not bind plasminogen at all. All three fusion proteins bound t-PA equivalently, providing the first evidence that CK18 may function as a t-PA receptor. t-PA and plasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen cannot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f still promoted plasminogen activation, probably reflecting the fact that CK8f was purified in dimeric or tetrameric form. These studies demonstrate that CK8 may promote plasminogen activation by t-PA only when present in an oligomerized state. CK18 may participate in the oligomer, together with CK8, based on its ability to bind t-PA.

Sympathetic activity : modulator of myocardial hypertrophy

The mechanisms regulating myocardial hypertrophy are largely unknown. Furthermore, the hypertrophic phenotype can be associated with either normal or abnormal function. To study the molecular mechanisms involved in myocardial hypertrophy, we have established a cell culture system in which stimulation of the alpha 1-adrenergic receptor leads to the development of myocardial cell hypertrophy. In addition to producing a generalized twofold increase in both cell size, total protein, and total RNA, activation of the alpha 1-receptor produces specific alterations in gene expression that are reflected by changes at both the mRNA and protein levels. In particular, alpha 1 stimulation leads to an increase in the expression of the c-myc oncogene as well as a selective increase in skeletal alpha-actin and beta-myosin heavy-chain isogene expression, isoforms normally found only in fetal/neonatal hearts. Similar changes in gene expression are seen in pressure-load hypertrophy in vivo. Skeletal alpha-actin gene expression is induced preferentially to that of the cardiac actin isogene resulting from a specific preferential increase in gene transcription. Work with subtype-specific inhibitors indicates that it is a particular alpha 1-receptor subtype that is responsible for the development of hypertrophy in culture. The finding that alpha 1 stimulation leads to an increase in protein kinase C activity is suggestive of a potential second messenger involving the phosphorylation of a transcriptional factor or factors.

Sympathetic modulation of the cardiac myocyte phenotype: studies with a cell-culture model of myocardial hypertrophy

Myocardial hypertrophy is the common endpoint of many cardiovascular stimuli such as hypertension, myocardial infarction, valvular disease, and congestive failure. Catecholamines have long been implicated in the pathogenesis of myocardial hypertrophy, however, it is very difficult to sort out catecholamine mechanisms in vivo. We have developed a cell-culture model which excludes hemodynamic effects and allows the assignment of receptor specificity to catecholamine effects. Utilizing this system, we have shown that stimulation of the alpha 1 adrenergic receptor leads to the development of myocardial hypertrophy and results in the selective up-regulation of the fetal/neonatal mRNAs encoding skeletal alpha-actin and beta-MHC, a pattern similar to that seen with hypertrophy in-vivo. Utilizing a co-transfection assay, we have also obtained data that suggest that the beta-PKC isozyme is in a pathway regulating transcription of the beta-MHC isogene. Beta adrenergic stimulation of the cultured cardiac myocytes also results in a modest degree of hypertrophy, however, this effect may be dependent upon myocyte contractile activity and may involve, at least in part, the non-muscle cells present in the culture system.

Epidermal growth factor differentially regulates low density lipoprotein receptor-related protein gene expression in neoplastic and fetal human astrocytes.

Low density lipoprotein receptor–related protein (LRP) is a multi‐functional endocytotic receptor that may modify the biological activity of reactive astrocytes in neuroplasticity and neurodegeneration and of malignant astrocytes in brain invasion. In this study, the regulation of LRP by epidermal growth factor receptor (EGFR) ligands in both cultured human fetal astrocytes and astrocytic tumor cell lines (U‐251 MG and U‐1242 MG) was investigated. All astrocytic cell types expressed LRP, as determined by the binding of activated α2‐macroglobulin (α2M*) on intact cells and by Western and Northern blot analyses of cell extracts. Primary cultured astrocytes expressed the highest levels of α2M*‐binding capacity (Bmax = 30 fmol/mg protein). This was twofold higher than for the U‐1242 MG astrocytoma cells (Bmax = 15 fmol/mg protein) and fourfold greater than for the glioblastoma U‐251 MG cells (7.0 fmol/mg protein). Receptor affinity (KD) ranged from 0.25 to 0.6 nM in all the astroglial cell types. Functional LRP at the surface was down‐regulated by EGF, compared with controls, as indicated by a reduction of both Bmax and LRP‐mediated endocytosis by approximately 50% and 60%, respectively. In comparison, EGF treatment of primary astrocytes did not down‐regulate LRP expression or LRP‐mediated endocytosis. Treatment of the tumor cells with EGF or TGFα (25 ng/ml) significantly down‐regulated total cellular LRP. Receptor‐associated protein (RAP) mRNA expression was not affected by EGF in either tumor cells or primary astrocytes. The reduction of LRP in the tumor cells resulted from a specific decrease in LRP mRNA transcription, as determined by Northern blot and nuclear run‐on experiments. These data suggest that EGF mediates a functional down‐regulation of LRP endocytotic activity in astrocytic tumor cells and that LRP expression is differentially regulated in neoplastic and non‐neoplastic astrocytes. GLIA 25:71–84, 1999.