Suppression of insulin-like3 receptor reveals the role of ß-catenin and Notch signaling in gubernaculum development.

During male development, the testes move from a high intraabdominal position and descend into the scrotum. The gubernaculum, an inguinoscrotal ligament connecting the testis to the lower abdomen, is believed to play a critical role in this process. The first stage of testicular descent is controlled by insulin like3 hormone (INSL3), produced in testicular Leydig cells. Deletion of Insl3 or its receptor, Rxfp2, in mice causes cryptorchidism. We produced Cre/loxP regulated shRNA transgenic mice targeting RXFP2 expression. We have shown that the transgene was able to reduce Rxfp2 gene expression and thus behaved as a hypomorphic allele of Rxfp2. Variable degrees of uni- and bilateral cryptorchidism was detected in males with the activated shRNA transgene on an Rxfp2+/- background. Conditional suppression of Rxfp2 in the gubernaculum led to cryptorchidism. Gene expression analysis of a mutant cremasteric sac using Illumina microarrays indicated abnormal expression of a significant number of genes in Wnt/ß-catenin and Notch pathways. We have demonstrated profound changes in the expression pattern of ß-catenin, Notch1, desmin, and androgen receptor (AR), in Rxfp2-/- male embryos, indicating the role of INSL3 in proliferation, differentiation, and survival of specific cellular components of the gubernaculum. We have shown that INSL3/RXFP2 signaling is essential for myogenic differentiation and maintenance of AR-positive cells in the gubernaculum. Males with the deletion of ß-catenin or Notch1 in the gubernacular ligament demonstrated abnormal development. Our data indicates that ß-catenin and Notch pathways are potential targets of INSL3 signaling during gubernacular development.

Suppression of relaxin receptor RXFP1 decreases prostate cancer growth and metastasis.

Relaxin (RLN) is a small peptide hormone expressed in several cancers of reproductive and endocrine organs. Increased expression of RLN in prostate cancer correlates with aggressive cancer. RLN G-protein-coupled receptor (RLN family peptide receptor 1, RXFP1) is expressed in both androgen receptor (AR)-positive and -negative prostate cancers as well as in prostate cancer cell lines. RLN behaves as a cell growth factor and increases invasiveness and proliferation of cancer cells in vitro and in vivo. The objective of this study is to determine whether downregulation of RXFP1 expression using small interfering RNA (siRNA) reduces cancer growth and metastasis in a xenograft model of prostate cancer. We used two well-characterized prostate adenocarcinoma cell lines, AR-positive LNCaP cells and AR-negative PC3 cells. The tumors were established in nude male mice by s.c. injections. Intratumoral injections of siRNAs loaded on biodegradable chitosan nanoparticles led to a downregulation of RXFP1 receptor expression and a dramatic reduction in tumor growth. In LNCaP tumors, the siRNA treatment led to an extensive necrosis. In PC3 xenografts treated with siRNA against RXFP1, the smaller tumor size was associated with the decreased cell proliferation and increased apoptosis. The downregulation of RXFP1 resulted in significant decrease in metastasis rate in PC3 tumors. Global transcriptional profiling of PC3 cells treated with RXFP1 siRNA revealed genes with significantly altered expression profiles previously shown to promote tumorigenesis, including the downregulation of MCAM, MUC1, ANGPTL4, GPI, and TSPAN8. Thus, the suppression of RLN/RXFP1 may have potential therapeutic benefits in prostate cancer.

INSL3 has tumor-promoting activity in thyroid cancer.

The functional role of INSL3 and its receptor RXFP2 in carcinogenesis is largely unknown. We have previously demonstrated (pro-)cathepsin-L as a target of INSL3 in human thyroid cancer cells facilitating penetration of tumor cells through elastin matrices. We demonstrate the expression of RXFP2 in human thyroid tissues and in mouse follicular thyroid epithelial cells using Cre-recombinase transgene driven by Rxfp2 promoter. Recombinant and secreted INSL3 increased the motility of thyroid carcinoma (TC) cells in an autocrine/paracrine manner. This effect required the presence of RXFP2. We identified S100A4 as a novel INSL3 target molecule and showed that S100A4 facilitated INSL3-induced enhanced motility. Stable transfectants of the human follicular TC cell line FTC-133 expressing and secreting bioactive human INSL3 displayed enhanced anchorage-independent growth in soft agar assays. Xenotransplant experiments in nude mice showed that INSL3, but not EGFP-mock transfectants, developed fast-growing and highly vascularized xenografts. We used human umbilical vein endothelial cells in capillary tube formation assays to demonstrate increased 2-dimensional tube formations induced by recombinant human INSL3 and human S100A4 comparable to the effect of vascular endothelial growth factor used as positive control. We conclude that INSL3 is a powerful and multifunctional promoter of tumor growth and angiogenesis in human thyroid cancer cell xenografts. INSL3 actions involve RXFP2 activation and the secretion of S100A4 and (pro-)cathepsin-L.

INSL3/RXFP2 signaling in testicular descent.

Mutations of the insulin-like peptide 3 (INSL3) hormone or its receptor, RXFP2, cause intraabdominal cryptorchidism in male mice. Specific RXFP2 expression in mouse gubernacula was detected at embryonic day 14.5 and markedly increased after birth in the developing cremaster muscle, as well as in the epididymis and testicular Leydig and germ cells. INSL3 treatment stimulated cell proliferation of embryonic gubernacular and Leydig cells, implicating active INSL3-mediated signaling. The transcription factor SOX9, a known male sex determination factor, upregulated the activity of the RXFP2 promoter. INSL3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10, although these factors are important for inguinoscrotal testicular descent. Similarly, conditional ablation of the androgen receptor gene in gubernacular cells resulted in disruption of inguinoscrotal descent. We performed mutation screening of INSL3 and RXFP2 in human patients with cryptorchidism and control subjects from different populations in Europe and the USA. Several missense mutations were described in both the INSL3 and RXFP2 genes. A novel V39G INSL3 mutation in a patient with cryptorchidism was identified; however, the functional analysis of the mutant peptide did not reveal compromised function. In more than 2000 patients and controls analyzed to date, the T222P RXFP2 mutation is the only one strongly associated with the mutant phenotype. The T222P mutant receptor, when transfected into 293T cells, had severely decreased cell membrane expression, providing the basis for the functional deficiency of this mutation.

Developmental expression and gene regulation of insulin-like 3 receptor RXFP2 in mouse male reproductive organs.

The mutations of testicular insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. Here we have examined Rxfp2 gene expression at different stages of embryonic and postnatal mouse development in male reproductive tissues employing quantitative RT-PCR and several RXFP2-specific antibodies directed toward different parts of the RXFP2 protein. Receptor expression was markedly increased after birth and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult mouse cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells and identifying TM3 as a novel in vitro model to study the effects of RXFP2 signaling. We generated Tg(Rxfp2-cre)Aia (Rxfp2-iCre) transgenic mice expressing improved Cre recombinase (iCre) under the control of the 2.4-kb mouse Rxfp2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive Rxfp2 gene expression during transabdominal testis descent. We demonstrated that the transcription factor Sox9, a known male sex determination factor, is expressed in mouse embryonic gubernacula and upregulated human, but not mouse, promoter luciferase reporter constructs. In conclusion, we have determined the developmental expression profile of INSL3 receptor employing newly characterized RXFP2 antisera and a novel Rxfp2-iCre transgenic mouse model. We determined the promoter region capable of providing the gubernacular-specific expression of Rxfp2. Analysis of RXFP2 promoter identified SOX9 as a new transcriptional enhancer of human gene expression.

T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane.

Insulin-like 3 (INSL3) hormone plays a crucial role in testicular descent during embryonic development. Genetic ablation of Insl3 or its G protein-coupled receptor (GPCR) Lgr8 causes cryptorchidism in mice. Previously, we identified a nonfunctional T222P mutation of LGR8 in several human patients with testicular maldescent. Using a large population of patients and healthy controls from Italy, we have demonstrated that T222P LGR8 mutation is present only in affected patients (19 T222P/+ of 598 vs. 0/450, P < 0.0001). We have also identified a novel allele of LGR8 (R223K) found in one patient with retractile testes. Both mutations are located in the leucine-rich repeats (LRRs) of GPCR ectodomain. The expression analysis of T222P mutant receptor transfected into 293T cells revealed that the mutation severely compromised GPCR cell membrane expression. The substitution of Thr(222) with the neutral Ser or Ala, or the R223K mutation, did not alter receptor cell membrane expression or ligand-induced cAMP increase. Additional mutations, affecting first leucine in a signature LxxLxLxxN/CxL stretch of LRR (L283F), or the amino acid residues, forming the disulfide bond or coordinating calcium ion in the LDLa module (C71Y and D70Y), also rendered proteins with reduced cell surface expression. The structural alterations of both LRRs and LDLa of the ligand-binding part of LGR8 cause the inability of receptor to express on the cell surface membrane and might be responsible for the abnormal testicular phenotype in patients.

Over expression of insulin-like 3 does not prevent cryptorchidism in GNRHR or HOXA10 deficient mice.

PURPOSE: Insulin-like 3 hormone is critical for the induction of growth and differentiation of gubernacular ligaments during embryonic testicular descent. Mice with mutation of insulin-like 3 or its receptor show high intra-abdominal cryptorchidism. We tested whether transgenic over expression of Insl3 can reverse inguinoscrotal cryptorchidism in mice deficient in Gnrhr or Hoxa10 genes. MATERIALS AND METHODS: Hoxa10 and Gnrhr deficient mice were intercrossed with Insl3 transgenic mice. The phenotype of the mutant mice and expression of the genes involved in testicular descent were analyzed. Using quantitative reverse transcriptase-polymerase chain reaction we evaluated expression of the genes in neonatal gubernacular cells on INSL3 (Phoenix Pharmaceuticals, Belmont, California) and testosterone stimulation. RESULTS: Transgenic over expression of Insl3 failed to restore normal testicular descent in Hoxa10 or Gnrhr deficient males. Histological evaluation did not reveal any differences in Insl3 transgenic gubernacula in either mutant. In mutant females Insl3 over expression resulted in transabdominal descent of the ovaries to the low abdominal position with the subsequent development of inguinal hernia. Expression of androgen receptor, insulin-like 3 receptor and Hoxa10 was not affected after incubation of neonatal gubernacular cells with insulin-like 3 or androgen. CONCLUSIONS: The results suggest that insulin-like 3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10 but their presence is important for inguinoscrotal testicular descent.

Endocrine effects of relaxin overexpression in mice.

Relaxin is a small peptide hormone with a variety of biological functions. To investigate the systemic endocrine effects of relaxin, we produced mice with transgenic overexpression of the Rln1 gene, Tg(Rln1), driven by rat insulin 2 promoter. The expression of relaxin was detected in the pancreas of the transgenic animals. An analysis of the sera from the transgenic animals revealed at least 20-fold elevation of the level of bioactive relaxin. Transgenic animals had normal viability and fertility in both sexes. Transgenic overexpression of Rln1 did not rescue the undescended testis phenotype in Insl3-deficient males, suggesting that in vivo relaxin does not interact with the insulin-like 3 factor receptor, leucine-rich repeats-containing G protein-coupled receptor 8, Lgr8. Phenotypically, the excess of relaxin resulted in hypertrophic nipple development in virgin female mice. Deletion of the relaxin receptor, leucine-rich repeats-containing G protein-coupled receptor 7, Lgr7, in Tg(Rln1) animals abrogated the development of enlarged nipples in females, indicating that relaxin exerts its effect through Lgr7 alone. The levels of previously defined targets of relaxin signaling, such as matrix metalloproteinases 2 and 9, vascular endothelial growth factor, or nitric oxide, were similar in the sera of the transgenic and wild-type mice. However, the total plasma protein concentration in male Tg(Rln1) mice was lower than that in control animals. The livers of male Tg(Rln1) mice exhibited significantly higher hydroxyproline content, indicative of increased collagen deposition. Our results indicate that relaxin overexpression causes gender-specific changes in liver collagen metabolism.

Genetic targeting of relaxin and insulin-like factor 3 receptors in mice.

Relaxin (RLN) is a small peptide hormone that affects a variety of biological processes. Rln1 knockout mice exhibit abnormal nipple development, prolonged parturition, agerelated pulmonary fibrosis, and abnormalities in the testes and prostate. We describe here RLN receptor Lgr7-deficient mice. Mutant females have grossly underdeveloped nipples and are unable to feed their progeny. Some Lgr7-/- females were unable to deliver their pups. Histological analysis of Lgr7 mutant lung tissues demonstrates increased collagen accumulation and fibrosis surrounding the bronchioles and the vascular bundles, absent in wild-type animals. However, Lgr7-deficient males do not exhibit abnormalities in the testes or prostate as seen in Rln1 knockout mice. Lgr7-deficient females with additional deletion of Lgr8 (Great), another putative receptor for RLN, are fertile and have normal-sized litters. Double mutant males have normal-sized prostate and testes, suggesting that Lgr8 does not account for differences in Rln1-/- and Lgr7-/- phenotypes. Transgenic overexpression of Insl3, the cognate ligand for Lgr8, does not rescue the mutant phenotype of Lgr7-deficient female mice indicating nonoverlapping functions of the two receptors. Our data indicate that neither Insl3 nor Lgr8 contribute to the RLN signaling pathway. We conclude that the Insl3/Lgr8 and Rln1/Lgr7 actions do not overlap in vivo.

Transabdominal testicular descent is disrupted in mice with deletion of insulinlike factor 3 receptor.

BACKGROUND: Several factors are implicated in transabdominal testicular descent, including insulinlike factor 3 (INSL3) hormone and Müllerian-inhibiting substance (MIS). A transgene insertional mutation found on chromosome 5 in the mouse, known as crsp, causes deletion of a transmembrane G protein-coupled receptor gene, Great, which is highly expressed in the gubernaculum. The authors describe here a detailed analysis of the testicular descent and gubernacular development in crsp mice to determine the role of the Great gene in this process. METHODS: Homozygous (crsp/crsp) mutant and wild-type heterozygous (crsp/+) mice were examined at birth (D 0) and at days 10 (D 10) and 30 (D 30) postnatally. Serial sagittal or coronal sections were analyzed for position of the gonads and cremaster sac development. RESULTS: Transabdominal testicular descent was absent at D 0 in crsp/crsp homozygous mice with no swelling reaction in the gubernacula. By D 10 the cremaster sac was significantly thinner (P <.05) and contained less collagen in the mutants than in the wild-type controls. On D 30 the cremaster sacs of mutant males were similar in thickness to those in females. CONCLUSIONS: Disruption of the Great gene causes failure of the transabdominal phase of descent, identical to that seen in the Insl3-deficient mutants, consistent with the recent data suggesting that Great gene encodes the Insl3 transmembrane receptor. No differences between D 30 mutant males and females were found in the gubernacula, suggesting that Insl3/Great signaling regulates gubernacular development.

GREAT/LGR8 is the only receptor for insulin-like 3 peptide.

During male development testes descend from their embryonic intraabdominal position into the scrotum. Two genes, encoding the insulin-like 3 peptide (INSL3) and the GREAT/LGR8 G protein-coupled receptor, control the differentiation of gubernaculum, the caudal genitoinguinal ligament critical for testicular descent. It was established that the INSL3 peptide activates GREAT/LGR8 receptor in vitro. Mutations of Insl3 or Great cause cryptorchidism (undescended testes) in mice. Overexpression of the transgenic Insl3 causes male-like gubernaculum differentiation, ovarian descent into lower abdominal position, and reduced fertility in females. To address the question whether Great deletion complements the mutant female phenotype caused by the Insl3 overexpression, we have produced Insl3 transgenic mice deficient for Great. Such females had a wild-type phenotype, demonstrating that Great was the only cognate receptor for Insl3 in vivo. We have established that pancreatic HIT cells, transfected with the INSL3 cDNA, produce functionally active peptide. Analysis of five INSL3 mutant variants detected in cryptorchid patients showed that P49S substitution renders functionally compromised peptide. Therefore, mutations in INSL3 might contribute to the etiology of cryptorchidism. We have also showed that synthetic insulin-like peptides (INSL4 and INSL6) were unable to activate LGR7 or GREAT/LGR8.

Isolation and expression analysis of the canine insulin-like factor 3 gene.

The insulin-like factor 3 (INSL3 or relaxin-like factor) is a hormone produced mainly in gonadal tissues in males and females. Deletion of INSL3 or its receptor in male mice leads to the undescended testes, or cryptorchidism. Here we describe an isolation and analysis of full-length canine INSL3 gene. The INSL3 gene is composed of two exons within a small genomic region. Putative translation of the isolated cDNA yields 132 amino acid preproINSL3 that has the domain structure characteristic for the insulin-relaxin peptide superfamily with a well-conserved receptor-binding domain. Northern blot hybridization showed stronger expression of INSL3 in testis than in ovary. Reverse transcription-polymerase chain reaction analysis of the INSL3 expression revealed a minor splice variant of INSL3 potentially encoding 105 amino acids peptide. We established that the medium, conditioned with recombinant canine INSL3, produced from the full-length cDNA, but not from the minor splice variant, activated human GREAT/LGR8 receptor in vitro. In addition to the functional allele of INSL3, genomic DNA of one of the analyzed dogs contained an intronless nonexpressed pseudogene of INSL3. We isolated canine INSL3 promoter and showed that its activity was strongly mediated by steroidogenic factor-1 in vitro. Using site-specific mutagenesis, we identified a well-conserved steroidogenic factor-1 binding site within canine INSL3 promoter.

Mutations of the GREAT gene cause cryptorchidism.

In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1-3% of newborn boys. The clinical consequences of this abnormality are infertility in adulthood and a significantly increased risk of testicular malignancy. Recently, we described a mouse transgene insertional mutation, crsp, causing high intraabdominal cryptorchidism in homozygous males. A candidate gene Great (G-protein-coupled receptor affecting testis descent), was identified within the transgene integration site. Great encodes a seven-transmembrane receptor with a close similarity to the glycoprotein hormone receptors. The Great gene is highly expressed in the gubernaculum, the ligament that controls testicular movement during development, and therefore may be responsible for mediating hormonal signals that affect testicular descent. Here we show that genetic targeting of the Great gene in mice causes infertile bilateral intraabdominal cryptorchidism. The mutant gubernaculae fail to differentiate, indicating that the Great gene controls their development. Mutation screening of the human GREAT gene was performed using DHPLC analysis of the genomic DNA from 60 cryptorchid patients. Nucleotide variations in GREAT cDNA were found in both the patient and the control populations. A unique missense mutation (T222P) in the ectodomain of the GREAT receptor was identified in one of the patients. This mutant receptor fails to respond to ligand stimulation, implicating the GREAT gene in the etiology in some cases of cryptorchidism in humans.