Genomic organization, expression profile, and characterization of the new protein PRA1 domain family, member 2 (PRAF2).

PRA1 domain family, member 2 (PRAF2) is a new 19 kDa protein with four putative transmembrane (TM) domains. PRAF2 (formerly designated JM4) belongs to a new protein family, which plays a role in the regulation of intracellular protein transport. Recently, PRAF2 was found to interact with the chemokine receptor CCR5. In order to further study the function and regulation of PRAF2, we determined its genomic structure and its protein expression pattern in normal and cancerous human tissues. PRAF2 encodes a 178-residue protein, whose sequence is related to PRAF1 (PRA1/prenylin) and PRAF3 (JWA/GTRAP3-18). The human PRAF2 gene contains three exons separated by two introns and is located on human chromosome Xp11.23. The recombinant PRAF2 protein was readily expressed in Schneider 2 (S2) insect cells, and the native protein was detected in human tissues with strong expression in the brain, small intestine, lung, spleen, and pancreas. The protein was undetectable in tissue of the testes. Strong PRAF2 protein expression was also found in human tumor tissues of the breast, colon, lung, and ovary, with a weaker staining pattern in normal tissues of the same patient. Our studies show for the first time that the CCR5-interacting PRAF2 protein is expressed in several human tissues with a possible function in ER/Golgi transport and vesicular traffic.

Upregulation of connexin 43 by retinoids but not by non-provitamin A carotenoids requires RARs.

Expression of connexin 43 (Cx43) is correlated with reduced indexes of neoplasia and is upregulated by cancer-preventive retinoids and carotenoids in nontransformed human and murine fibroblasts and keratinocytes. The molecular mechanism of upregulation, however, is poorly understood. Three retinoic acid receptor (RAR) antagonists (Ro 41-5253, BMS453, and BMS493) were capable of suppressing retinoid-induced Cx43 protein expression in 10T1/2 cells. However, Ro 41-5253 did not suppress protein expression by the non-provitamin A carotenoids astaxanthin or lycopene. In contrast, Cx43 induction by astaxanthin but not by a RAR-specific retinoid was inhibited by GW9662, an antagonist of peroxisome proliferator activated receptor-gamma activation. Simultaneous treatment with the maximally effective concentration of a retinoid and with beta-carotene or the non-provitamin A carotenoid astaxanthin resulted in supraadditive upregulation of Cx43 expression, again indicating separate mechanisms of gene regulation by these two cancer preventive agents.

Cancer prevention by retinoids and carotenoids: independent action on a common target.

Virtually all human tumors are deficient in gap junctional communication (GJC) and the restoration of GJC by forced expression of connexins reduces indices of neoplasia. The expression of connexin 43 (Cx43) is upregulated by cancer-preventive retinoids and carotenoids which correlates with the suppression of carcinogen-induced transformation in 10T1/2 cells. However, the molecular mechanism for upregulated expression is poorly understood. The retinoic acid receptor antagonist, Ro 41-5253, suppressed retinoid-induced Cx43 protein expression in 10T1/2 cells and the induction of a Cx43 luciferase reporter construct in F9 cells, but did not suppress protein expression or reporter activity induced by the non-pro-vitamin A carotenoid astaxanthin. In contrast, Cx43 induction by astaxanthin, but not by a RAR-specific retinoid, was inhibited by GW9662, a PPAR-gamma antagonist. Neither compound required protein synthesis for the induction of Cx43 mRNA, nor was the 5.0 h half-life of Cx43 mRNA altered, indicating direct transcriptional activation. The responsive region was found within -158 bp and +209 bp of the transcription start site. Site directed mutagenesis of a GC-box in this region increased basal levels of transcription and loss of retinoid responsiveness. Simultaneous treatment with a retinoid and beta-carotene or astaxanthin resulted in supra-additive Cx43 expression, again indicating separate mechanisms of gene regulation.

Transcriptional regulation of connexin 43 expression by retinoids and carotenoids: similarities and differences.

Gap junctions, connexons, are formed by assembly of trans-membrane connexin proteins and have multiple functions including the coordination of cell responses. Most human tumors are deficient in gap junctional communication (GJC) and restoration of GJC by forced expression of connexins reduces indices of neoplasia. Expression of connexin 43 (Cx43), the most widely-expressed connexin family member, is upregulated by cancer-preventive retinoids and carotenoids in normal and preneoplastic cells; an action considered of mechanistic significance. However, the molecular mechanism for upregulated expression is poorly understood. The retinoic acid receptor antagonist Ro 41-5253 was capable of suppressing retinoid-induction Cx43 luciferase reporter construct in F9 cells, but did not suppress reporter activity induced by the non-pro-vitamin A carotenoids astaxanthin or lycopene, indicating that retinoids have separate mechanisms of gene activation than non-pro-vitamin A carotenoids. Neither class of compound required protein synthesis for induction of Cx43 mRNA, nor was the 5.0 h half-life of Cx43 mRNA altered, indicating direct transcriptional activation. The responsive region was found within -158 bp and +209 bp of the transcription start site; this contains a Sp1/Sp3 GC-box to which Sp1 and Sp3 were bound, as revealed by electrophoretic mobility shift assays (EMSA), but no retinoic acid response element (RARE). Site directed mutagenesis of this GC-box resulted in increased basal levels of transcription and loss of responsiveness to a synthetic retinoid. In this construct astaxanthin and lycopene produced marginally, but not significantly higher, reporter activity than the control.

Cancer chemoprevention by connexins.

Gap junctions, or connexons, are formed by connexin proteins and connect most cells in the body to form water-filled channels directly linking the cytoplasm. Among the molecules known to be transferred via junctions are cAMP, ATP, IP3 and glucose. Tumor cells are in general deficient in functional gap junctions either as a result of gene silencing, or failure to correctly process and assemble connexons. Tumor promoters inhibit function whereas certain cancer preventive agents increase junctional communication. When connexin expression in tumor cells is forced by introduction of exogenous genes or is increased by pharmacological agents, connexin expression reduces growth in suspension and growth as xenografts in nude mice. It is as yet unclear if in tumor cells these actions depend on junctional transfer of signal molecules or reflect some other function of these genes. Restoration of connexin function offers an exciting opportunity to delay tumor progression and inhibit metastasis.