This study has examined the expression of two new facets of the growth hormone axis, the growth hormone secretagogue receptor (GHS-R) and its recently identified putative natural ligand ghrelin, in prostate cancer cells. GHS-R 1a and 1b isoforms and ghrelin mRNA expression were detected by RT-PCR in the ALVA-41, LNCaP, DU145 and PC3 prostate cancer cell lines. A normal prostate cDNA library expressed GHS-R1a, but not the 1b isoform or ghrelin. Immunohistochemical staining for the GHS-R 1a isoform and ghrelin was positive in the four cell lines studied. PC3 cells showed increased cell proliferation in vitro in response to ghrelin to levels 33% above untreated controls, implying a potential tumourpromoting role for ghrelin in this tissue. This study is the first to demonstrate the co-expression of the GHS-R and ghrelin in prostate cancer cells. It is also the first study to provide evidence that a previously unrecognised autocrine/paracrine pathway involving ghrelin, that is capable of stimulating growth, exists in prostate cancer.

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), has been primarily linked to the central neuroendocrine regulation of GH secretion and food intake, although additional peripheral actions of ghrelin have also been reported. In this context, the expression of ghrelin and its cognate receptor has been recently demonstrated in rat testis, suggesting a role for this molecule in the direct control of male gonadal function. However, whether this signaling system is present in human testis remains largely unexplored. In this study we report the expression and cellular location of ghrelin and its functional receptor, the type 1a GHS-R, in adult human testis. In addition, evaluation of ghrelin and GHS-R1a immunoreactivity in testicular tumors and dysgenetic tissue is presented. The expression of the mRNAs encoding ghrelin and GHS-R1a was demonstrated in human testis specimens by RT-PCR, followed by direct sequencing. In normal testis, ghrelin immunostaining was demonstrated in interstitial Leydig cells and, at lower intensity, in Sertoli cells within the seminiferous tubules. In contrast, ghrelin was not detected in germ cells at any stage of spermatogenesis. The cognate ghrelin receptor showed a wider pattern of cellular distribution, with detectable GHS-R1a protein in germ cells, mainly in pachytene spermatocytes, as well as in somatic Sertoli and Leydig cells. Ghrelin immunoreactivity was absent in poorly differentiated Leydig cell tumor, which retained the expression of GHS-R1a peptide. In contrast, highly differentiated Leydig cell tumors expressed both the ligand and the receptor. The expression of ghrelin and GHS-R1a was also detected in dysgenetic Sertoli cell-only seminiferous tubules, whereas germ cell tumors (seminoma and embryonal carcinoma) were negative for ghrelin and were weakly positive for GHS-R1a. In conclusion, our results demonstrate that ghrelin and the type 1a GHS-R are expressed in adult human testis and testicular tumors. Overall, the expression of ghrelin and its functional receptor in human and rat testis, with roughly similar patterns of cellular distribution, is highly suggestive of a conserved role for this newly discovered molecule in the regulation of mammalian testicular function.

Ghrelin is a novel 28-amino acid peptide identified as the endogenous ligand for the GH secretagogue receptor (GHS-R). Besides its hallmark central neuroendocrine effects in the control of GH secretion and food intake, an unexpected reproductive facet of ghrelin has recently emerged because expression of this molecule and its cognate receptor has been demonstrated in rat testis. However, whether this signaling system is present in human gonads remains to be evaluated. In this study, we have assessed the presence and cellular location of ghrelin and its functional receptor, namely the type 1a GHS-R, in the cyclic human ovary by means of immunohistochemistry using specific polyclonal antibodies. Strong ghrelin immunostaining was demonstrated in ovarian hilus interstitial cells. In contrast, ghrelin signal was not detected in ovarian follicles at any developmental stage, nor was it present in newly formed corpora lutea (CL) at very early development. However, specific ghrelin immunoreactivity was clearly observed in young and mature CL, whereas expression of the peptide disappeared in regressing luteal tissue. Concerning the cognate receptor, ovarian expression of GHS-R1a protein showed a wider pattern of tissue distribution, with detectable specific signal in oocytes as well as somatic follicular cells; luteal cells from young, mature, old, and regressing CL; and interstitial hilus cells. Of particular note, follicular GHS-R1a peptide expression paralleled follicle development with stronger immunostaining in granulosa and theca layers of healthy antral follicles. In conclusion, our results are the first to demonstrate that ghrelin and its functional type 1a receptor are expressed in the cyclic human ovary with distinct patterns of cellular location. The presence of both components (ligand and receptor) of the ghrelin signaling system within the human ovary opens up the possibility of a potential regulatory role of this novel molecule in ovarian function under physiological and pathophysiological conditions.

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epitheliallike cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelialmesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.