Jeffrey R. Leipprandt

Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

Both estrogen (E) and progesterone (P) are implicated in the etiology of human breast cancer. Defining their mechanisms of action, particularly in vivo, is relevant to the prevention and therapy of breast cancer. We investigated the molecular and cellular mechanisms of E and/or P-induced in vivo proliferation, in the normal rat mammary gland and in hormone-dependent rat mammary cancers which share many characteristics with the normal human breast and hormone-dependent breast cancers. We show that E+P treatment induced significantly greater proliferation in both the normal gland and mammary cancers compared to E alone. In both the normal gland and tumors, E+P-induced proliferation was mediated through the increased production of amphiregulin (Areg), an epidermal growth factor receptor (EGFR) ligand, and the activation of intracellular signaling pathways (Erk, Akt, JNK) downstream of EGFR that regulate proliferation. In vitro experiments using rat primary mammary organoids or T47D breast cancer cells confirmed that Areg and the synthetic progestin, R5020, synergize to promote cell proliferation through EGFR signaling. Iressa, an EGFR inhibitor, effectively blocked this proliferation. These results indicate that mediators of cross talk between E, P, and EGFR pathways may be considered as relevant molecular targets for the therapy of hormone-dependent breast cancers, especially in premenopausal women.

Amphiregulin mediates progesterone-induced mammary ductal development during puberty

IntroductionPuberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined.MethodsPubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL).ResultsP, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression.ConclusionsThese results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Progesterone receptor A-regulated gene expression in mammary organoid cultures.

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

Molecular defect of caprine N-acetylglucosamine-6-sulphatase deficiency. A single base substitution creates a stop codon in the 5'-region of the coding sequence.

Sanfilippo D syndrome (mucopolysaccharidosis type IIID, MPS IIID; McKusick 252940) is caused by the deficiency of N-acetylglucosamine-6-sulphatase ( (G6S; EC 3.1.6.14). A Nubian goat with this lysosomal storage disease has been identified (Thompson et al 1992). Towards further development of this animal model of MPS IIID, determination of the DNA defect was initiated. Southern analysis of restriction enzyme-digested goat genomic DNA indicated that there was no apparent deletion or rearrangement of G6S in the affected animal using both human and goat probes. Initial RT-PCR studies of RNA from affected animals indicated that G6S mRNA was present. For mutation analysis, a series of normal and affected caprine G6S RT-PCR amplicons, covering the entire coding region, was sequenced and compared. Either oligo(dT) or antisense primers were used for cDNA synthesis. Amplicons, 300400 bp long, were produced with primers based on caprine G6S clone sequences or human sequence (Robertson et al 1992). A nonsense mutation was found in the cDNA of the affected animal, changing a C to T in codon 102 of the 559-amino-acid G6S coding sequence. This mutation, in addition to truncating the protein, also introduces an AluI restriction site (underlined below) (recognition sequence AGCT) that will provide a convenient basis for carrier detection. Validation of a PCR-based test is now underway.

Identification of a bovine β-mannosidosis mutation and detection of two β-mannosidase pseudogenes

Abstract.β-Mannosidase deficiency results in β-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine β-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3′ end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for β-mannosidosis carrier screening. Application of this test also revealed the presence of two β-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (>99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine β-mannosidosis phenotype suggests that the 22 C-terminal amino acids of β-mannosidase play an important role in the function of this enzyme.

CLONING AND SEQUENCE ANALYSIS OF CAPRINE N-ACETYLGLUCOSAMINE 6-SULFATASE CDNA

Mucopolysaccharidosis IIID results from the deficiency of N-acetylglucosamine 6-sulfatase activity. A Nubian goat with this lysosomal storage disease has been identified. As a first step in developing this animal model for testing treatment methods, we cloned and sequenced the caprine N-acetylglucosamine 6-sulfatase cDNA coding region. Overall there is 88% nucleotide homology between the goat and human sequence and 94% homology of the deduced amino acid sequence. The human and two ruminant species differ by the presence of an imperfect trinucleotide (CCG) repeat in the ruminant signal sequence.

Topography of the Prostaglandin Endoperoxide H2 Synthase-2 in Membranes

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe66 and Leu67, were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe59, Phe66, Tyr76, and Phe84 to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg62 and Lys64, with the phospholipid head groups.

In utero hematopoietic stem cell transplantation: a caprine model for prenatal therapy in inherited metabolic diseases.

Objectives: We explored the feasibility and efficacy of in utero hematopoietic stem cell transplantation in the caprine animal model system with the objectives of determining procedures for transplantation and establishing methods for detecting engraftment. Methods: Male fetal liver hematopoietic stem cells were injected into female fetuses during the immunotolerant period, using either hysterotomy or ultrasound-guided injections. Results: The rate of fetal death was much lower for the ultrasound-guided injections. Donor cells were observed in the peritoneal fluid of 4 fetuses 3 days after injection, but no donor cells were detected in tissues at longer time periods. Conclusions: Ultrasound-guided injection of hematopoietic stem cells into the abdomen of a developing fetus is safe and feasible. The parameters required for successful engraftment have not yet been identified.

Epidermal Growth Factor Receptor (EGFR) Signaling Is a Key Mediator of Hormone-Induced Leukocyte Infiltration in the Pubertal Female Mammary Gland

It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor κB ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland.

Determination of Genotypic Frequency of Caprine Mucopolysaccharidosis IIID

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases caused by enzyme deficiencies in the degradative pathways of glycosaminoglycans (GAGs). Because of a specific enzyme deficiency, GAGs or partially degraded GAGs accumulate in the lysosomes and may be excreted in the urine. The 4 types of MPS III (Sanfilippo syndromes A–D), characterized by the inability to degrade heparan sulfate (HS), are clinically similar, but significant phenotypic variation is observed within and among MPS III subtypes.5 Mucopolysaccharidosis IIID (McKusick 252940) is caused by a deficiency in N-acetylglucosamine 6-sulfatase activity (G6S; EC 3.1.6.14). Clinical, biochemical, morphological, and immunohistochemical characterization of human and caprine MPS IIID has been reported.3,8 Heparan sulfate and N-acetylglucosamine 6-sulfate accumulate in the tissues and urine of individuals affected by MPS IIID.8 A secondary storage of gangliosides also occurs in the central nervous system. Caprine G6S cDNA has been cloned and sequenced,2 and the cDNA defect in caprine MPS IIID has been determined.1 A mutation test utilizing the polymerase chain reaction (PCR) has been established for carrier detection and prenatal screening for caprine G6S deficiency.4 Using the mutation test, 552 purebred Nubian goats (52 males and 500 females) from 20 herds in central lower Michigan (herd sizes of 1–210 goats and ages ranging from newborn to 10 years) were surveyed to determine the frequencies of the homozygous and heterozygous genotypes for MPS IIID. Efficacies of 2 DNA sampling techniques, collection of buccal epithelial cells (BECs) on cytology brushesa (a noninvasive procedure used for human PCR diagnosis6) and collection of white blood cells (WBC), were also compared. BECs were collected by swirling a cytology brush on the buccal mucosa of the lower lip. The brush was rinsed in 200 ml 50 mM NaOH. Cells were lysed by boiling for 10 minutes and then neutralized with 10 ml 2 M Tris-HCl, pH 8.0. Blood was collected in ethylene diaminetetraacetic acid (EDTA) collection tubes.b WBCs were isolated by combining 100 ml whole blood and 0.5 ml red cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.4), incubating the solution on ice for 10 minutes, centrifuging at 12,000 3 g for 30 seconds, and removing the supernatant. Cells were lysed as above. The previously described G6S PCR-based mutation test4 was used with the following minor modifications. One-microliter aliquots of the lysed cells were used as the DNA