Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells

P75/AIRM1 is a recently identified surface molecule that belongs to the sialoadhesin family and displays homology with the myeloid cell antigen CD33. In lymphoid cells, p75/AIRM1 is confined to natural killer cells and mediates inhibition of their cytolytic activity. In this study, we show that p75/AIRM1 is also expressed by cells of the myelomonocytic cell lineage, in which it appears at a later stage as compared with CD33. In vitro proliferation and differentiation of cord blood-derived CD34+ cells (induced by stem cell factor and granulocyte–macrophage colony-stimulating factor) were consistently inhibited by the addition of anti-p75/AIRM1 mAb. Engagement of CD33 led to inhibition in some experiments. A sharp decrease of cell proliferation/survival was detected in all three p75/AIRM1+ chronic myeloid leukemias analyzed when cultured in the presence of either anti-p75/AIRM1 or anti-CD33 mAbs. Thus, the present study suggests that p75/AIRM1 and CD33 may play a regulatory role in normal myelopoiesis and may be viewed as suitable target molecules to counteract the proliferation/survival of chronic myeloid leukemias.

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

Primary Mesenchymal Cells Isolated from SPARC-null Mice Exhibit Altered Morphology and Rates of Proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

Involvement of protein kinase A in fibroblast growth factor-2-activated transcription

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Epistatic and independent functions of Caspase-3 and Bcl-XL in developmental programmed cell death

The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-XL prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-XL directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x/caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-XL in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-XL in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets.

Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.

Androgen-induced proliferative quiescence in prostate cancer cells: The role of AS3 as its mediator

In the prostate gland of adult mammals, most epithelial cells are in a state of proliferative quiescence. Androgens regulate this effect by inducing cell cycle arrest in the G0/G1 phase. Potential mediators of this androgen-induced proliferative shutoff were identified by means of subtracted cDNA libraries. The expression pattern of one of these sequences, AS3, strongly correlated with the expression of the androgen-induced proliferative shutoff both temporally and dosewise. The AS3 gene is located on chromosome 13 q12.3, in close proximity to the BRCA2 gene. The loss of chromosomal regions where AS3 alleles are located correlates with various human cancers, including prostate. The biological effect of AS3 was tested in two stable cell lines, one expressing sense and another expressing antisense AS3 constructs, both under tetracycline regulation. S9 cells were obtained by retroviral infection with virions containing a tetracycline-regulated sense AS3 construct. In these cells, sense AS3 was negatively regulated by tetracycline. Tetracycline withdrawal increased the expression of AS3 mRNA and protein. The expression of tetracycline-regulated AS3 resulted in inhibition of cell proliferation. A4 cells were obtained by retroviral infection with virions containing a tetracycline-regulated antisense AS3 construct. Vector-driven expression of antisense-AS3 blocked the induction of androgen-induced endogenous AS3 mRNA and blocked the inhibitory effect of androgens on cell proliferation. Tetracycline-regulated expression of the empty vector control had no effect on cell proliferation. These experiments strongly suggest that AS3 is a mediator of the androgen-induced proliferative shutoff.

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Inflammatory mediators are perpetuated in macrophages resistant to apoptosis induced by hypoxia

A hypoxic/anoxic microenvironment has been proposed to exist within a vascular lesion due to intimal or medial cell proliferation in vascular diseases. Here, we examined whether hypoxia alters macrophage function by exposing murine macrophage-like RAW 264.7 (RAW) cells to hypoxia (2% O2). When cells were exposed to hypoxia, a significant number of RAW cells underwent apoptosis. Additionally, small subpopulations of RAW cells were resistant to hypoxia-induced apoptosis. Through repeated cycles of hypoxia exposure, hypoxia-induced apoptosis-resistant macrophages (HARMs) were selected; HARM cells demonstrate >70% resistance to hypoxia-induced apoptosis, as compared with the parental RAW cells. When heat shock protein (HSP) expression was examined after hypoxia, we observed a significant decrease in constitutive heat shock protein 70 (HSC 70) in RAW cells, but not in HARMs, as compared with the control normoxic condition (21% O2). In contrast, the expression level of glucose-regulated protein 78 (GRP 78) in RAW and HARM cells after hypoxia treatment was not altered, suggesting that HSC 70 and not GRP 78 may play a role in protection against hypoxia-induced apoptosis. When tumor necrosis factor α (TNF-α) production was examined after hypoxic treatment, a significant increase in TNF-α production in HARM but decrease in RAW was observed, as compared with cells cultured in normoxic conditions. HARM cells also exhibit a much lower level of modified-LDL uptake than do RAW cells, suggesting that HARMs may not transform into foam cells. These results suggest that a selective population of macrophages may adapt to potentially pathological hypoxic conditions by overcoming the apoptotic signal.

Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)

The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-α-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2−/− T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2−/− T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.