Back-to-Germline (B2G) Procedure for Antibody Devolution.

Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described 'back-to-germline' (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are 'de-matured' to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6, two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4flox/flox ; Gata6flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (Hsd3b1, Cyp17a1 and Hsd17b3) was reduced, whereas expression of another Leydig cell marker, Insl3, was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2.

GATA4 Regulates Blood-Testis Barrier Function and Lactate Metabolism in Mouse Sertoli Cells.

Conditional deletion of Gata4 in Sertoli cells (SCs) of adult mice has been shown to increase permeability of the blood-testis barrier (BTB) and disrupt spermatogenesis. To gain insight into the molecular underpinnings of these phenotypic abnormalities, we assessed the impact of Gata4 gene silencing in cell culture models. Microarray hybridization identified genes dysregulated by siRNA-mediated inhibition of Gata4 in TM4 cells, an immortalized mouse SC line. Differentially expressed genes were validated by quantitative RT-PCR analysis of primary cultures of Gata4(flox/flox) mouse SCs that had been subjected to cre-mediated recombination in vitro. Depletion of GATA4 in TM4 cells and primary SCs was associated with altered expression of genes involved in key facets of BTB maintenance, including tight/adherens junction formation (Tjp1, Cldn12, Vcl, Tnc, Csk) and extracellular matrix reorganization (Lamc1, Col4a1, Col4a5, Mmp10, Mmp23, Timp2). Western blotting and immunocytochemistry demonstrated reduced levels of tight junction protein-1, a prototypical tight junction protein, in GATA4-depleted cells. These changes were accompanied by a loss of morphologically recognizable junctional complexes and a decline in epithelial membrane resistance. Furthermore, Gata4 gene silencing was associated with altered expression of Hk1, Gpi1, Pfkp, Pgam1, Gls2, Pdk3, Pkd4, and Ldhb, genes regulating the production of lactate, a key nutrient that SCs provide to developing germ cells. Comprehensive metabolomic profiling demonstrated impaired lactate production in GATA4-deficient SCs. We conclude that GATA4 plays a pivotal role in the regulation of BTB function and lactate metabolism in mouse SCs.

GATA4 is a key regulator of steroidogenesis and glycolysis in mouse Leydig cells.

Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells. The role of GATA4 in Leydig cell development, however, remains controversial, because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses. We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are down-regulated or up-regulated after small interfering RNA (siRNA)-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1. These same genes were dysregulated when primary cultures of Gata4(flox/flox) adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Among the down-regulated genes were enzymes of the androgen biosynthetic pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a). Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed alteration of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, Gpi1, Pfkp, and Pgam1), lower intracellular levels of ATP, and increased extracellular levels of glucose. Our findings suggest that GATA4 plays a pivotal role in Leydig cell function and provide novel insights into metabolic regulation in this cell type.

Novel markers of gonadectomy-induced adrenocortical neoplasia in the mouse and ferret.

Gonadectomy (GDX) induces sex steroid-producing adrenocortical tumors in certain mouse strains and in the domestic ferret. Transcriptome analysis and DNA methylation mapping were used to identify novel genetic and epigenetic markers of GDX-induced adrenocortical neoplasia in female DBA/2J mice. Markers were validated using a combination of laser capture microdissection, quantitative RT-PCR, in situ hybridization, and immunohistochemistry. Microarray expression profiling of whole adrenal mRNA from ovariectomized vs. intact mice demonstrated selective upregulation of gonadal-like genes including Spinlw1 and Insl3 in GDX-induced adrenocortical tumors of the mouse. A complementary candidate gene approach identified Foxl2 as another gonadal-like marker expressed in GDX-induced neoplasms of the mouse and ferret. That both "male-specific" (Spinlw1) and "female-specific" (Foxl2) markers were identified is noteworthy and implies that the neoplasms exhibit mixed characteristics of male and female gonadal somatic cells. Genome-wide methylation analysis showed that two genes with hypomethylated promoters, Igfbp6 and Foxs1, are upregulated in GDX-induced adrenocortical neoplasms. These new genetic and epigenetic markers may prove useful for studies of steroidogenic cell development and for diagnostic testing.

Toying with fate: Redirecting the differentiation of adrenocortical progenitor cells into gonadal-like tissue.

Cell fate decisions are integral to zonation and remodeling of the adrenal cortex. Animal models exhibiting ectopic differentiation of gonadal-like cells in the adrenal cortex can shed light on the molecular mechanisms regulating steroidogenic cell fate. In one such model, prepubertal gonadectomy (GDX) of mice triggers the formation of adrenocortical neoplasms that resemble luteinized ovarian stroma. Transcriptomic analysis and genome-wide DNA methylation mapping have identified genetic and epigenetic markers of GDX-induced adrenocortical neoplasia. Members of the GATA transcription factor family have emerged as key regulators of cell fate in this model. Expression of Gata4 is pivotal for the accumulation of gonadal-like cells in the adrenal glands of gonadectomized mice, whereas expression of Gata6 limits the spontaneous and GDX-induced differentiation of gonadal-like cells in the adrenal cortex. Additionally, Gata6 is essential for proper development of the adrenal X-zone, a layer analogous to the fetal zone of the human adrenal cortex. The relevance of these observations to developmental signaling pathways in the adrenal cortex, to other animal models of altered adrenocortical cell fate, and to human diseases is discussed.

Expression of Wnt and TGF-ß pathway components and key adrenal transcription factors in adrenocortical tumors: association to carcinoma aggressiveness.

Factors controlling benign and malignant adrenocortical tumorigenesis are largely unknown, but several mouse models suggest an important role for inhibin-alpha (INHA). To show that findings in the mouse are relevant to human tumors and clinical outcome, we investigated the expression of signaling proteins and transcription factors involved in the regulation of INHA in human tumor samples⋅ Thirty-one adrenocortical tumor samples, including 13 adrenocortical carcinomas (ACCs), were categorized according to Weiss score, hormonal profile, and patient survival data and analyzed using immunohistochemistry and RT-PCR. Expression of the TGF-ß signaling mediator SMAD3 varied inversely with Weiss score, so that SMAD3 expression was lowest in the most malignant tumors. By contrast, SMAD2 expression was upregulated in most malignant tumors. Wnt pathway co-receptors LRP5 and LRP6 were predominantly expressed in benign adrenocortical tumors. In ACCs, expression of transcription factors GATA-6 and SF-1 correlated with that of their target gene INHA. Moreover, the diminished expression of GATA-6 and SF-1 in ACCs correlated with poor outcome. We conclude that the factors driving INHA expression are reduced in ACCs with poor outcome, implicating a role for INHA as a tumor suppressor in humans.

Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse.

DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine-guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.

Conditional mutagenesis of Gata6 in SF1-positive cells causes gonadal-like differentiation in the adrenal cortex of mice.

Transcription factor GATA6 is expressed in the fetal and adult adrenal cortex and has been implicated in steroidogenesis. To characterize the role of transcription factor GATA6 in adrenocortical development and function, we generated mice in which Gata6 was conditionally deleted using Cre-LoxP recombination with Sf1-cre. The adrenal glands of adult Gata6 conditional knockout (cKO) mice were small and had a thin cortex. Cytomegalic changes were evident in fetal and adult cKO adrenal glands, and chromaffin cells were ectopically located at the periphery of the glands. Corticosterone secretion in response to exogenous ACTH was blunted in cKO mice. Spindle-shaped cells expressing Gata4, a marker of gonadal stroma, accumulated in the adrenal subcapsule of Gata6 cKO mice. RNA analysis demonstrated the concomitant upregulation of other gonadal-like markers, including Amhr2, in the cKO adrenal glands, suggesting that GATA6 inhibits the spontaneous differentiation of adrenocortical stem/progenitor cells into gonadal-like cells. Lhcgr and Cyp17 were overexpressed in the adrenal glands of gonadectomized cKO vs control mice, implying that GATA6 also limits sex steroidogenic cell differentiation in response to the hormonal changes that accompany gonadectomy. Nulliparous female and orchiectomized male Gata6 cKO mice lacked an adrenal X-zone. Microarray hybridization identified Pik3c2g as a novel X-zone marker that is downregulated in the adrenal glands of these mice. Our findings offer genetic proof that GATA6 regulates the differentiation of steroidogenic progenitors into adrenocortical cells.

Expression and localization of claudins-3 and -12 in transformed human brain endothelium.

BACKGROUND: The aim of this study was to characterize the hCMEC/D3 cell line, an in vitro model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12. FINDINGS: hCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function in vitro and also recommended for routine hCMEC/D3 culture. CONCLUSIONS: These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics in vitro.

GATA4 is a critical regulator of gonadectomy-induced adrenocortical tumorigenesis in mice.

In response to gonadectomy certain inbred mouse strains develop sex steroidogenic adrenocortical neoplasms. One of the hallmarks of neoplastic transformation is expression of GATA4, a transcription factor normally present in gonadal but not adrenal steroidogenic cells of the adult mouse. To show that GATA4 directly modulates adrenocortical tumorigenesis and is not merely a marker of gonadal-like differentiation in the neoplasms, we studied mice with germline or conditional loss-of-function mutations in the Gata4 gene. Germline Gata4 haploinsufficiency was associated with attenuated tumor growth and reduced expression of sex steroidogenic genes in the adrenal glands of ovariectomized B6D2F1 and B6AF1 mice. At 12 months after ovariectomy, wild-type B6D2F1 mice had biochemical and histological evidence of adrenocortical estrogen production, whereas Gata4(+/-) B6D2F1 mice did not. Germline Gata4 haploinsufficiency exacerbated the secondary phenotype of postovariectomy obesity in B6D2F1 mice, presumably by limiting ectopic estrogen production in the adrenal glands. Amhr2-cre-mediated deletion of floxed Gata4 (Gata4(F)) in nascent adrenocortical neoplasms of ovariectomized B6.129 mice reduced tumor growth and the expression of gonadal-like markers in a Gata4(F) dose-dependent manner. We conclude that GATA4 is a key modifier of gonadectomy-induced adrenocortical neoplasia, postovariectomy obesity, and sex steroidogenic cell differentiation.