Tight junction composition is altered in the epithelium of polycystic kidneys.

Kidney cysts in autosomal dominant polycystic kidney disease (ADPKD) undergo progressive enlargement together with luminal fluid secretion. This involves active, uphill transcellular Cl(-) transport which drives passive Na(+) and water secretion. Implicit in this mechanism is the assumption that the paracellular permeability of the cyst epithelium to Cl(-) must be very low. Claudins are tight junction (TJ) transmembrane proteins that determine the ion selectivity of paracellular barriers. The aim of this study was to determine the expression and localization of claudins within renal cysts in a mouse hypomorphic model of ADPKD and in human patients. We found that the majority of cysts were of collecting duct origin. Claudins normally expressed in collecting duct (3, 4, 7, 8, and 10) were found in small cysts. However, only claudin-7 persisted at substantive levels in the dedifferentiated epithelium of large, presumably late-stage cysts, where it was localized both at the TJ and basolaterally. The constitutively expressed TJ proteins, ZO-1 and occludin, were also abundantly expressed and correctly localized, suggesting that the basic infrastructure of the TJ is preserved. A previous study suggested that claudin-7 may function as a paracellular Cl(-) barrier. We postulate that the role of claudin-7 in ADPKD is to seal the paracellular route in Cl(-)-secreting cyst epithelium, preventing backleak of Cl(-), and that it thereby plays a permissive role in fluid secretion and cyst growth.

Growth hormone gene expression in normal lymph nodes and lymphomas of the dog.

Growth hormone (GH) is involved in the development, maturation, and function of the immune system. Recent studies have demonstrated that GH can be synthesized and secreted by lymphoid tissues, where it may act as an autocrine/paracrine growth factor. To determine whether GH may be involved in the development of hematological malignancies, GH gene expression in canine lymphomas was investigated. GH mRNA was detected in non-tumorous lymph nodes and in the majority of the lymphomas, by RT-PCR analysis. In situ and Northern blot hybridizations were negative. Analysis of the transcriptional start sites of the GH gene using 5'-RACE (rapid amplification of cDNA ends) showed that the canine lymphoid transcripts contained a 33-85 bp enlarged 5'-untranslated region compared to the pituitary and mammary GH transcripts. Part of the lymphoid GH transcripts contained intron 1, which would result in early termination of the translation due to an in-frame stopcodon. GH measurements in lymphoid tissues revealed a low content of immunoreactive GH. The results presented demonstrate that canine lymphoid tissue is an extrapituitary site of GH gene expression. However, GH production appeared to be low, indicating that lymphoid GH is probably not a major factor in the development or progression of canine lymphoma.

Cloning, characterization, and physical mapping of the canine Prop-1 gene (PROP1): exclusion as a candidate for combined pituitary hormone deficiency in German shepherd dogs.

Abnormalities in the genes encoding Pit-1 and Prop-1 have been reported to cause combined pituitary hormone deficiency (CPHD) in mice and humans. In dogs, a similar phenotype has been described in the German shepherd breed. We have previously reported that the Pit-1 gene (POU1F1) is not mutated in affected German shepherd dogs. In this study, we report the isolation and mapping of the canine Prop-1 gene (PROP1), and we assessed the involvement of PROP1 in German shepherd dog dwarfism. The canine PROP1 gene was found to contain three exons, encoding a 226 amino acid protein. The deduced amino acid sequence was 79% and 84% homologous with the mouse and human Prop-1 protein, respectively. Using fluorescence in situ hybridization, PROP1 was mapped to canine chromosome 11. Further mapping with a canine radiation hybrid panel showed co-localization with the polymorphic DNA marker AHT137. Sequence analysis of genomic DNA from dwarf German shepherd dogs revealed no alterations in the PROP1 gene. Moreover, linkage analysis of AHT137 revealed no co-segregation between the PROP1 locus and the CPHD phenotype, excluding this gene as candidate for canine CPHD and providing a new spontaneous model of hypopituitarism.

Cloning and cellular localization of the canine progesterone receptor: co-localization with growth hormone in the mammary gland.

The mammary gland has been found to express the gene encoding growth hormone (GH) in several species. Within the mammary gland, it may act as an autocrine/paracrine growth factor for cyclic epithelial changes, and may be a determinant in mammary carcinogenesis. In the dog, progestins enhance mammary GH expression. To elucidate the mechanism of progestin-induced mammary GH expression, the canine progesterone receptor (PR) is characterized and the cellular localization of the PR in normal and tumorous mammary tissues is examined. Sequence analysis of the canine PR revealed two in-frame ATG codons, encoding a putative PR-B protein of 939 amino acids and a putative PR-A protein of 765 amino acids. Western blot analysis indicated that both isoforms occur in uterus and mammary gland issues. Immunohistochemical analysis of the PR revealed that the PR was differentially expressed in mammary tissue, with many PR-positive epithelial cells in the proliferation phase of the glandular tissue and a low number of PR-positive cells in differentiated mammary tissue. Stromal and myoepithelial cells had no specific PR staining. Mammary tumours had a variety of staining patterns, including no staining, normal nuclear staining, marked heterogeneous immunoreactivity and perinuclear staining of tumorous epithelial cells and cytoplasmic-staining of spindle cells. Double staining showed that all GH-producing cells were positive for PR, whereas not all PR containing cells stained for GH. It is concluded that the activated PR may transactivate GH expression in the mammary gland within the same cell and functions as a pre-requisite transcription factor. However, during malignant transformation this regulation may be lost.

Cloning of the canine gene encoding transcription factor Pit-1 and its exclusion as candidate gene in a canine model of pituitary dwarfism.

Combined pituitary hormone deficiency (CPHD) is an autosomal recessive inherited disease of German shepherd dogs characterized primarily by dwarfism. In mice and humans a similar genetic disorder has been described that results from an alteration in the gene encoding the transcription factor Pit-1. In this study we characterized the canine Pit-1 gene, determined the chromosomal localization of the Pit-1 gene, and screened dwarf German shepherd dogs for the presence of mutations in this gene. The full-length canine Pit-1 cDNA contained an open reading frame encoding 291 amino acids, 92 bp of 5'-untranslated region, and 1959 bp of 3'-untranslated region. The deduced amino acid sequence was highly homologous with Pit-1 of other mammalian species. Using a Pit-1 BAC clone as probe, the Pit-1 gene was mapped by FISH to canine Chromosome (Chr) 31. In dwarf German shepherd dogs a C to A transversion was detected, causing a Phe (TTC) to Leu (TTA) substitution at codon 81. This alteration was present neither in other canine breeds analyzed nor in other mammalian species. However, healthy German shepherd dogs were also homozygous for the mutant allele, indicating that it is not the primary disease-causing mutation. In addition, linkage analysis of polymorphic DNA markers flanking the Pit-1 gene, 41K19 and 52L05, revealed no co-segregation between the Pit-1 locus and the CPHD phenotype. These findings suggest that a gene other than Pit-1 is responsible for the pituitary anomaly in dwarf German shepherd dogs.

Mammary growth hormone and tumorigenesis--lessons from the dog.

The discovery in the early 1990s that progestin-induced growth hormone (GH) excess in the dog originates in the mammary gland can be seen as a hallmark in the research on the pathogenesis of mammary cancer in the dog. The local biosynthesis and release of GH may provide a highly proliferative environment in the mammary gland, which contributes to the development and/or progression of mammary tumours. Before final goals such as prevention of tumour formation or inhibition of tumour promotion can be achieved it is of eminent importance to elucidate the mechanism of progesterone-induced mammary GH production and the mechanism of local autocrine/paracrine action of GH. These local GH effects may be achieved through direct growth stimulating effects of GH as well as by indirect effects mediated by the stimulation of the biosynthesis of insulin-like growth factor-I (IGF-I). The biological effects of the IGFs largely depend on the presence of IGF binding proteins (IGFBPs) which may both enhance or inhibit the activity of the IGFs. This review concentrates on recent advances in the understanding of the local mammary GH-IGF axis and the lessons which can be drawn from the dog for mammary cancer research in other species.

Canine mammary growth hormone gene transcription initiates at the pituitary-specific start site in the absence of Pit-1.

Growth hormone (GH) gene expression has been reported in the mammary glands of various mammalian species. The mechanism by which the GH gene becomes activated in extrapituitary tissues is currently unclear. We have characterized the canine mammary and pituitary GH gene transcripts by Northern blot, 5'- and 3'-RACE (rapid amplification of cDNA ends), and DNA sequence analysis. Northern blot analysis detected GH gene transcripts in mammary glands of dogs which were exposed to high levels of progestins. The mammary and pituitary GH cDNAs were shown to be identical in both the coding region and untranslated regions. Pituitary GH gene expression is highly dependent upon the transcription factor Pit-1. Analysis of Pit-1 gene expression using RT-PCR followed by Southern hybridization revealed a strong pituitary signal but faint, weak or no hybridization signals in mammary gland samples. Among the negative samples were progestin-treated dogs with high mammary GH gene expression. These findings indicate that mammary and pituitary GH gene transcripts originate from the same transcription start site but are regulated differentially.