Prostate stromal and urogenital sinus mesenchymal cell lines for investigations of stromal-epithelial interactions

Abstract Bidirectional signaling between the urogenital sinus epithelium and mesenchyme is an essential element of prostate development that regulates ductal morphogenesis, growth, and differentiation. Comparable interactions between the epithelium and stroma in the adult prostate appear to regulate normal growth homeostasis. Alterations in the stromal–epithelial dialogue that recapitulate features of the mesenchymal–epithelial interactions of development may play a critical role in the development of benign prostatic hyperplasia and in the progression of prostate cancer. For this reason, the mesenchymal–epithelial interactions of development are of considerable interest. In this review, we provide an overview of the mesenchymal contribution to rodent prostate development with an emphasis on the stage just before ductal budding (embryonic day 16; E16) and describe the isolation, characterization and utility of a newly established E16 urogenital sinus mesenchymal cell line.     

Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate

The budding of the urogenital sinus epithelium into the surrounding mesenchyme signals the onset of prostate morphogenesis. The epithelial and mesenchymal factors that regulate ductal budding and the ensuing process of ductal growth and branching are not fully known. We provide evidence that bone morphogenetic protein 4 (BMP4) is a mesenchymal factor that regulates ductal morphogenesis. The Bmp4 gene was most highly expressed in the male urogenital sinus from embryonic day 14 through birth, a period marked by formation of main prostatic ducts and initiation of ductal branching. From an initial wide distribution throughout the prostatic anlage of the urogenital sinus, Bmp4 expression became progressively restricted to the mesenchyme immediately surrounding the nascent prostatic ducts and branches. Exogenous BMP4 inhibited epithelial cell proliferation and exhibited a dose-dependent inhibition of ductal budding in urogenital sinus tissues cultured in vitro. Adult Bmp4 haploinsufficient mice exhibited an increased number of duct tips in both the ventral prostate and coagulating gland. Taken together, our data indicate that BMP4 is a urogenital sinus mesenchymal factor that restricts prostate ductal budding and branching morphogenesis.     

LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs

Lens epithelium–derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors.     

Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.     

Anchorage-independent culture maintains prostate stem cells

Freshly isolated mouse prostate epithelial cells regenerate fully differentiated prostate tissue when combined with embryonic urogenital sinus mesenchyme and grafted in vivo. We show here that this regenerative capacity, which has been attributed to a small population of pleuripotential progenitor epithelial cells, is rapidly lost when the cells are placed in monolayer culture but can be maintained by culture in anchorage-independent conditions. Epithelial cells placed in anchorage-independent culture formed proliferating spheres that could be serially passaged and exhibited increased expression of putative stem cell markers as compared to cells grown in monolayer culture. Epithelial cells isolated from the fetal urogenital sinus, the newborn, and adult prostate formed spheres with similar efficiency, while cells isolated from the post-castration prostate exhibited significantly higher sphere-forming abilities. When passaged spheres were recombined with E17 rat urogenital sinus mesenchyme and grafted in vivo, they generated fully differentiated mouse prostate glandular epithelium containing both p63+ basal cells and p63− luminal cells and expressing a variety of prostate-specific and terminal differentiation markers.     

Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys substitutions by Mn2+ in vitro

The D,DX(35)E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg(2+) or Mn(2+)), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg(2+) and Mn(2+), and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX(35)E motif. Mn(2+) but not Mg(2+) can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn(2+) only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex.