Increased expression of peripheral benzodiazepine receptor (PBR) in dimethylbenz[a]anthracene-induced mammary tumors in rats

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B_max of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B_max of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.     

The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA.

A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.     

Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression

High levels of peripheral-type benzodiazepine receptor (PBR), the alternative-binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non-tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon-rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

Transient protection by peripheral benzodiazepine receptors during the early events of ultraviolet light-induced apoptosis.

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in the formation of mitochondrial permeability transition (PT) pores which play a critical role during the early events of apoptosis. PBRs are located in many tissues and are strongly expressed in the superficial layers of human epidermis. PBRs play a protective role against free radical damage and PBR ligands modulate apoptosis. To investigate the role of PBR during the early events of ultraviolet (UV)-mediated apoptosis we compared the effects of UVB on PBR-transfected Jurkat cells and their wild type counterparts devoid of any PBR expression. Results indicate that early after UVB exposure (up to 4 h), PBR-transfected cells were more resistant to apoptosis and exhibited a delayed mitochondrial transmembrane potential drop, a diminished superoxide anions production, and a reduced caspase-3 activation. Taken together these findings suggest that PBR may regulate early death signals leading to UV induced apoptosis.     

Expression of Peripheral Benzodiazepine Receptor (PBR) in Human Tumors: Relationship to Breast,Colorectal, and Prostate Tumor Progression

Abstract

High levels of peripheral‐type benzodiazepine receptor (PBR), the alternative‐binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non‐tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon‐rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

PAP7, a PBR/PKA-RIalpha-associated protein: a new element in the relay of the hormonal induction of steroidogenesis

The precise mechanism by which the hormone-induced minimal cAMP levels act at the mitochondria to activate cholesterol transport and steroid synthesis is unknown. We propose that this mechanism involves a macromolecular signaling complex where a newly identified peripheral-type benzodiazepine receptor (PBR)-associated protein (PAP7) binds the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA), thus allowing for local efficient catalytic activation and phosphorylation of the substrate steroidogenesis acute regulatory protein (StAR), leading to cholesterol transfer from the low affinity StAR to the high affinity PBR cholesterol binding protein. The mouse and human PAP7 proteins were cloned, their genomic organization and chromosomal localization characterized, their tissue distribution evaluated and subcellular localization defined. PAP7 is highly expressed in steroidogenic tissues, where it follows the pattern of PKA-RIalpha expression and data from a human adrenal disease suggest that it participates in PKA-RIalpha-mediated tumorigenesis and hormone-independent hypercortisolism. PAP7 is localized in the Golgi and mitochondria and inhibition of PAP7 expression results in reduced hormone-induced cholesterol transport into mitochondria and decreased steroid formation. Taken together, these data suggest that PAP7 functions as an A-kinase anchoring protein (AKAP) critical in the cAMP-dependent steroid formation.     

Design, synthesis, and structure-activity relationships of novel tetracyclic compounds as peripheral benzodiazepine receptor ligands

The peripheral benzodiazepine receptor (PBR) is pharmacologically distinct from the central benzodiazepine receptor (CBR) and has been identified in a wide range of peripheral tissues as well as in the central nervous system. Although numerous studies have been performed of it, the physiological roles and functions of the PBR are still unclear. In the present study, in exploring new types of ligands for PBR, we found that a new series of compounds having a tetracyclic ring system, which were designed from FGIN-1-27, exhibited high affinities for PBR. We prepared and evaluated them for PBR affinities. The results of binding tests showed that 12e and 12f were the most potent PBR ligands among them (12e: IC(50)=0.44nM, 12f: IC(50)=0.37nM). In this paper, we present the design, synthesis, and structure-activity relationships (SARs) of novel tetracyclic compounds.     

PAP7, a PBR/PKA-RIα-associated protein: a new element in the relay of the hormonal induction of steroidogenesis

The precise mechanism by which the hormone-induced minimal cAMP levels act at the mitochondria to activate cholesterol transport and steroid synthesis is unknown. We propose that this mechanism involves a macromolecular signaling complex where a newly identified peripheral-type benzodiazepine receptor (PBR)-associated protein (PAP7) binds the regulatory subunit RIα of the cAMP-dependent protein kinase A (PKA), thus allowing for local efficient catalytic activation and phosphorylation of the substrate steroidogenesis acute regulatory protein (StAR), leading to cholesterol transfer from the low affinity StAR to the high affinity PBR cholesterol binding protein. The mouse and human PAP7 proteins were cloned, their genomic organization and chromosomal localization characterized, their tissue distribution evaluated and subcellular localization defined. PAP7 is highly expressed in steroidogenic tissues, where it follows the pattern of PKA-RIα expression and data from a human adrenal disease suggest that it participates in PKA-RIα-mediated tumorigenesis and hormone-independent hypercortisolism. PAP7 is localized in the Golgi and mitochondria and inhibition of PAP7 expression results in reduced hormone-induced cholesterol transport into mitochondria and decreased steroid formation. Taken together, these data suggest that PAP7 functions as an A-kinase anchoring protein (AKAP) critical in the cAMP-dependent steroid formation.     

The lysosomal trafficking regulator interacting protein-5 localizes mainly in epithelial cells

Endocytosis, subsequent protein sorting into multivesicular bodies (MVBs), and eventual degradation in lysosomes compose an important mechanism for controlling protein expression on the plasma membrane. The lysosomal trafficking regulator interacting protein-5 (LIP5) is part of the complex protein machinery involved in MVB biosynthesis. LIP5 interacts with other players of the ESCRT machinery as well as with two known cargo proteins, AQP2 and EGFR, whose degradation is affected upon reduction of LIP5 expression. To investigate the expression and localization pattern of LIP5, we studied LIP5 protein expression in a mouse tissue panel and subjected various rodent and human tissues to immunohistochemistry. Immunoblotting revealed that, except for jejunum, LIP5 is expressed as a 42 kDa protein in all mouse tissues tested. Alternatively-spliced gene products could not be detected. Immunohistochemical studies revealed that in tissues positive for LIP5, LIP5 is detected in virtually all epithelial cells of the examined rodent and human tissues. The observed LIP5 expression in epithelial tissues suggests that LIP5 is of particular importance in the MVB sorting and degradation of proteins expressed in polarized cells.     

The repertoire of solute carriers of family 6: identification of new human and rodent genes

Tremendous amount of primary sequence information has been made available from the genome sequencing projects, although a complete annotation and identification of all genes is still far from being complete. Here, we present the identification of two new human genes from the pharmacologically important family of transporter proteins, solute carriers family 6 (SLC6). These were named SLC6A17 and SLC6A18 by HUGO. The human repertoire of SLC6 proteins now consists of 19 functional members and four pseudogenes. We also identified the corresponding orthologues and additional genes from mouse and rat genomes. Detailed phylogenetic analysis of the entire family of SLC6 proteins in mammals shows that this family can be divided into four subgroups. We used Hidden Markov Models for these subgroups and identified in total 430 unique SLC6 proteins from 10 animal, one plant, two fungi, and 196 bacterial genomes. It is evident that SLC6 proteins are present in both animals and bacteria, and that three of the four subfamilies of mammalian SLC6 proteins are present in Caenorhabditis elegans, showing that these subfamilies are evolutionary very ancient. Moreover, we performed tissue localization studies on the entire family of SLC6 proteins on a panel of 15 rat tissues and further, the expression of three of the new genes was studied using quantitative real-time PCR showing expression in multiple central and peripheral tissues. This paper presents an overall overview of the gene repertoire of the SLC6 gene family and its expression profile in rats.     

The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting

The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.     

The human SRY protein is present in fetal and adult Sertoli cells and germ cells

Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.     

Distribution of the human intracellular serpin protease inhibitor 8 in human tissues.

Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.     

Peripheral benzodiazepine receptors and mitochondrial function

For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.     

Global gene expression during the human organogenesis: from transcription profiles to function predictions

Human embryogenesis includes an integrated set of complex yet coordinated development of different organs and tissues, which is regulated by the spatiotemporal expression of many genes. Deciphering the gene regulation profile is essential for understanding the molecular basis of human embryo development. While molecular and genetic studies in mouse have served as a valuable tool to understand mammalian development, significant differences exists in human and mouse development at morphological and genomic levels. Thus it is important to carry out research directly on human embryonic development. Here we will review some recent studies on gene regulation during human embryogenesis with particular focus on the period of organogenesis, which had not been well studied previously. We will highlight a gene expression database of human embryos from the 4(th) to the 9(th) week. The analysis of gene regulation during this period reveals that genes functioning in a given developmental process tend to be coordinately regulated during human embryogenesis. This feature allows us to use this database to identify new genes important for a particular developmental process/pathway and deduce the potential function of a novel gene during organogenesis. Such a gene expression atlas should serve as an important resource for molecular study of human development and pathogenesis.