Journal of Cellular Biochemistry

Classical insulin and IGF‐1 receptors are α₂β₂ heterotetrameric complexes synthesized from two identical αβ half‐receptor precursors [1,2]. Recent data strongly suggests, however, that nonidentical αβ half‐receptor precursors can assemble to generate hybrid holoreceptor species both in vivo and in vitro [3–6,41]. This review focuses primarily on two types of hybrid receptors. The first type is an insulin/IGF‐1 hybrid receptor generated by the association of an αβ insulin half‐receptor with an αβ IGF‐1 half‐receptor. The second type is one formed from a wildtype (kinase‐active) insulin or IGF‐1 αβ half‐receptor and a mutant (kinase‐inactive) insulin αβ half‐receptor. Although the functional properties of insulin/IGF‐1 hybrid receptors have not yet been completely defined, wildtype/mutant hybrid receptors are essentially substrate kinase inactive [6]. These data indicate that the mutant αβ half‐receptor exerts a transdominant inhibition upon the wildtype αβ half‐receptor within the α₂β₂ holoreceptor complex. This defect in substrate kinase activity may contribute to the molecular defect underlying some syndromes of severe insulin resistance and diabetes. Heterozygous individuals expressing both wildtype and mutant tyrosine kinase‐defective insulin receptor precursors demonstrate varying degrees of insulin resistance and diabetes [7–11]. In addition, cell lines which express both endogenous wildtype and transfected kinase‐defective insulin receptors display markedly decreased insulin and IGF‐1 sensitivity and responsiveness [12–14]. Formation of hybrid receptors which results in premature termination of insulin signal transduction may be one mechanism underlying the observation that kinase‐inactive receptors inhibit the function of native receptors.

Perturbations in the balanced process of osteoblast‐mediated bone formation and osteoclast‐mediated bone resorption leading to excessive osteoclast formation and/or activity is the cause of many pathological bone conditions such as osteoporosis. The osteoclast is the only cell in the body capable of resorbing and degrading the mineralized bone matrix. Osteoclast formation from monocytic precursors is governed by the actions of two key cytokines macrophage‐colony‐stimulating factor and receptor activator of nuclear factor‐κB ligand (RANKL). Binding of RANKL binding to receptor RANK initiates a series of downstream signaling responses leading to monocytic cell differentiation and fusion, and subsequent mature osteoclast bone resorption and survival. The phosphoinositide‐3‐kinase (PI3K)‐protein kinase B (Akt) signaling cascade is one such pathway activated in response to RANKL. The 3‐phosphoinositide‐dependent protein kinase 1 (PDK1), is considered the master upstream lipid kinase of the PI3K‐Akt cascade. PDK1 functions to phosphorylate and partially activate Akt, triggering the activation of downstream effectors. However, the role of PDK1 in osteoclasts has yet to be clearly defined. In this study, we specifically deleted the PDK1 gene in osteoclasts using the cathepsin‐K promoter driven Cre‐LoxP system. We found that the specific genetic ablation of PDK1 in osteoclasts leads to an osteoclast‐poor osteopetrotic phenotype in mice. In vitro cellular assays further confirmed the impairment of osteoclast formation in response to RANKL by PDK1‐deficient bone marrow macrophage (BMM) precursor cells. PDK1‐deficient BMMs exhibited reduced ability to reorganize actin cytoskeleton to form a podosomal actin belt as a result of diminished capacity to fuse into giant multinucleated osteoclasts. Notably, biochemical analyses showed that PDK1 deficiency attenuated the phosphorylation of Akt and downstream effector GSK3β, and reduced induction of NFATc1. GSK3β is a reported negative regulator of NFATc1. GSK3β activity is inhibited by Akt‐dependent phosphorylation. Thus, our data provide clear genetic and mechanistic insights into the important role for PDK1 in osteoclasts.

Zinc is a trace element that is essential for the normal function of cells. It is a cofactor for the structure and function of a wide range of cellular proteins including enzymes, transcription factors, and structural proteins. Recent studies have shown that zinc plays a role in the development of various cancers. Unfortunately no established common relationships of zinc with cancer development and progression have been identified. Zinc is known to have systemic effects such as regulation of the immune system as well as direct cellular effects resulting in regulation of gene expression, bioenergetics, metabolic pathways, signal transduction and cell invasion. Zinc is also reported to regulate cell proliferation and growth. In this review presentation we focus on the effects of zinc that are involved in the regulation of apoptosis in malignant cells. We selected the apoptotic effects of zinc because zinc is reported to both induce apoptosis in some cancers and to protect other cancer cells against apoptosis induced by other factors. The effects of zinc in the regulation of apoptosis appear to be cell type specific. More importantly the reported effects of zinc on cancer cells must be viewed from the perspective of the physiological regulation of zinc homeostasis. Thus one must be mindful of the experimental conditions under which zinc effects are investigated relative to the physiological and pathological conditions of cellular zinc distribution and concentrations that can exist in situ. J. Cell. Biochem. 106: 750–757, 2009. © 2009 Wiley‐Liss, Inc.

The reactive metabolites of benzo[a]pyrene (B[a]P) and cyclopenta[c,d]pyrene (CPP) induced an accumulation/phosphorylation of p53 in Hepa1c1c7 cells, whereas inhibition of p53 reduced the apoptosis. Judged by the inhibiting effect of wortmannin, phosphatidyl‐inositol‐3 (PI‐3) kinases such as DNA‐dependent protein kinase (DNA‐PK), ATM (ataxia‐telangiectasia mutated), and/or ATR (ATM related kinase), appeared to be involved in the DNA damage recognition and the B[a]P‐/CPP‐induced accumulation of p53. B[a]P and CPP also induced phosphorylation of jun‐N‐terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). While inhibition of JNK had no effects on the B[a]P‐/CPP‐induced apoptosis, inhibition of p38 MAPK activity reduced this effect. Interestingly, survival signals such as phosphorylation of Akt and Bad seemed to be induced by the B[a]P‐/CPP‐compounds. Furthermore, also extracellular signal‐regulated kinase (ERK)1/2 was activated and seemed to function as a survival signal in B[a]P‐/CPP‐induced apoptosis. © 2004 Wiley‐Liss, Inc.

Hypoxic injury to the ocular surface provokes an inflammatory response that is mediated, in part, by corneal epithelial‐derived 12‐hydroxyeicosanoids. Recent studies indicate that a cytochrome P450 (CYP) monooxygenase, identified as CYP4B1, is involved in the production of these eicosanoids which exhibit potent inflammatory and angiogenic properties. We have isolated and cloned a corneal epithelial CYP4B1 full‐length cDNA and demonstrated that the CYP4B1 mRNA is induced by hypoxia in vitro and in vivo. To further understand the molecular regulation that underlies the synthesis of these potent inflammatory eicosanoids in response to hypoxic injury, we isolated and cloned the CYP4B1 promoter region. GenomeWalker libraries constructed from rabbit corneal epithelial genomic DNA were used as templates for primary and nested PCR amplifications with gene‐ and adaptor‐specific primers. A 3.41‐kb DNA fragment of the 5′‐flanking region of the CYP4B1 promoter was isolated, cloned, sequenced, and analyzed by computer software for the presence of knowncis‐acting elements. Analysis of the promoter sequence revealed the presence of consensus DNA binding sequences for factors known to activate gene transcription in response to hypoxia including HIF‐1, NFκB, and AP‐1. Transient transfection of luciferase reporter (pGL3‐Basic) vectors containing different lengths of the CYP4B1 promoter fragment demonstrated hypoxia‐induced transcription in rabbit corneal epithelial (RCE) cells. Electrophoretic mobility shift assay (EMSA) revealed a marked induction of nuclear binding activity for the labeled HIF‐1 probe from the CYP4B1 promoter in nuclear extracts of cells exposed to hypoxia. This binding activity was due to sequence‐specific binding to the HIF‐1 oligonucleotide probe as shown by competition with excess unlabeled probe for the HIF‐1 but not with unlabeled NFκB probe. The nuclear binding activity of AP‐1 and NFκB probes from the CYP4B1 promoter was also enhanced in response to hypoxia suggesting that these transcription factors contribute to the hypoxic induction of CYP4B1 expression. The results of this study provide the first molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids in response to hypoxic injury. Further studies are needed to fully evaluate the molecular regulation of this gene during inflammation. © 2004 Wiley‐Liss, Inc.

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor‐κB ligand (RANKL) in the presence of macrophage colony‐stimulating factor (M‐CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c‐derived cultures, osteoclast formation in both systems—osteoblast/bone‐marrow cell co‐cultures and in RANKL‐induced osteoclastogenesis—was inhibited by antibody to tumor necrosis factor‐α (TNF‐α), and was enhanced by the addition of this cytokine. TNF‐α itself promoted osteoclastogenesis in the presence of M‐CSF. However, even at high concentrations of TNF‐α the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF‐α mRNA and induced TNF‐α release from osteoclast progenitors. Furthermore, antibody to p55 TNF‐α receptors (TNF receptors‐1) (but not to p75 TNF‐α receptors (TNF receptors‐2) inhibited effectively RANKL‐ (and TNF‐α() induced osteoclastogenesis. Anti‐TNF receptors‐1 antibody failed to inhibit osteoclastogenesis in C57BL/6‐derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF‐α is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis. © 2001 Wiley‐Liss, Inc.

We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T‐antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178–1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M‐FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1–2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2–3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non‐transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro. J. Cell. Biochem. 87: 9–15, 2002. © 2002 Wiley‐Liss, Inc.

Ryanodine receptors (RyR) are involved in regulating intracellular Ca⁺⁺ mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR‐selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor‐mediated activation events and cytokine‐dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)‐2 by T lymphocytes responding to mitogenic anti‐CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation‐induced expression of the α chain (CD25) of the high affinity IL‐2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL‐2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL‐2‐dependent proliferation of CTLL‐2 T cells. These data indicate that RyR are involved in regulating IL‐2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR‐associated Ca⁺⁺ signaling regulates T cell proliferation by promoting both IL‐2 synthesis and IL‐2‐dependent cell cycle progression. © 2004 Wiley‐Liss, Inc.

In intestine, 24,25(OH)₂D₃, which is made under conditions of calcium‐, phosphate‐, and 1,25(OH)₂D₃ sufficiency, inhibits the stimulatory actions of 1,25(OH)₂D₃ on phosphate and calcium absorption. In the current work, we provide evidence that 24,25(OH)₂D₃‐mediated signal transduction occurs mechanistically through increased H₂O₂ production which involves binding of 24,25(OH)₂D₃ to catalase and resultant decreases in enzyme activity. Physiological levels of H₂O₂ mimicked the action of 24,25(OH)₂D₃ on inhibiting 1,25(OH)₂D₃‐stimulated phosphate uptake in isolated enterocytes. Moreover, the molecular basis of such inhibition was suggested by the presence of two thioredoxin domains in the 1,25D₃‐MARRS protein/ERp57: Exposure of cells to either 24,25(OH)₂D₃ or H₂O₂ gradually reduced 1,25(OH)₂D₃ binding to 1,25D₃‐MARRS protein, between 10 and 20 min of incubation, but not to VDR. Feeding studies with diets enriched in the antioxidants vitamins C and E showed that net phosphate absorption in vivo nearly doubled relative to chicks on control diet. Antioxidant diets also resulted in increased [³H]1,25(OH)₂D₃ binding to both 1,25D₃‐MARRS and VDR, suggesting benefits to both transcription‐ and membrane‐initiated signaling pathways. Intriguingly, phosphorous content of bones from birds on antioxidant diets was reduced, suggesting increased osteoclast activity. Because mature osteoclasts lack VDR, we analyzed a clonal osteoclast cell line by RT‐PCR and found it contained the 1,25D₃‐MARRS mRNA. The combined data provide mechanistic details for the 1,25(OH)₂D₃/24,25(OH)₂D₃ endocrine system, and point to a role for the 1,25D₃‐MARRS protein as a redox‐sensitive mediator of osteoclast activity and potential therapeutic target. J. Cell. Biochem. 99: 1572–1581, 2006. © 2006 Wiley‐Liss, Inc.

Cancer is a disease that claims millions of lives each year across the world. Despite advancement in technologies and therapeutics for treating the disease, these modes are often found to turn ineffective during the course of treatment. The resistance against drugs in cancer patients stems from multiple factors, which constitute genetic heterogeneity like gene mutations, tumor microenvironment, exosomes, miRNAs, high rate of drug efflux from cells, and so on. This review attempts to collate all such known and reported factors that influence cancer drug resistance and may help researchers with information that might be useful in developing better therapeutics in near future to enable better management of several cancers across the world.