Anti-Müllerian hormone and letrozole levels in boys with constitutional delay of growth and puberty treated with letrozole or testosterone.

STUDY QUESTION: Does treatment of constitutional delay of growth and puberty (CDGP) in boys with aromatase inhibitor letrozole (Lz) or conventional low-dose testosterone (T) have differing effects on developing seminiferous epithelium? SUMMARY ANSWER: Anti-Müllerian hormone (AMH) declined similarly in both treatment groups, and the two Sertoli cell-derived markers (AMH and inhibin B (iB)) exhibited differing responses to changes in gonadotrophin milieu. WHAT IS KNOWN ALREADY: Boys with CDGP may benefit from puberty-inducing medication. Peroral Lz activates gonadotrophin secretion, whereas intramuscular low-dose T may transiently suppress gonadotrophins and iB. STUDY DESIGN, SIZE, DURATION: Sera of 28 boys with CDGP who participated in a randomised, controlled, open-label trial at four paediatric centres in Finland between August 2013 and January 2017 were analysed. The patients were randomly assigned to receive either Lz (2.5 mg/day) (n = 15) or T (1 mg/kg/month) (n = 13) for 6 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 28 patients were at least 14 years of age, showed first signs of puberty, wanted medical attention for CDGP and were evaluated at 0, 3, 6 and 12 months of visits. AMH levels were measured with an electrochemiluminescence immunoassay and Lz levels with liquid chromatography coupled with tandem mass spectrometry. MAIN RESULTS AND THE ROLE OF CHANCE: AMH levels decreased in both treatment groups during the 12-month follow-up (P < 0.0001). Between 0 and 3 months, the changes in gonadotrophin levels (increase in the Lz group, decrease in the T group) correlated strongly with the changes in levels of iB (FSH vs iB, r = 0.55, P = 0.002; LH vs iB, r = 0.72, P < 0.0001), but not with the changes in AMH (P = NS). At 12 months, AMH levels did not differ between the groups (P = NS). Serum Lz levels (range, 124-1262 nmol/L) were largely explained by the Lz dose per weight (at 3 months r = 0.62, P = 0.01; at 6 months r = 0.52, P = 0.05). Lz levels did not associate with changes in indices of hypothalamic-pituitary-gonadal axis activity or Sertoli cell markers (in all, P = NS). LIMITATIONS, REASONS FOR CAUTION: The original trial was not blinded for practical reasons and included a limited number of participants. WIDER IMPLICATIONS OF THE FINDINGS: In early puberty, treatment-induced gonadotrophin stimulus was unable to counteract the androgen-mediated decrease in AMH, while changes in iB levels were associated with changes in gonadotrophin levels. AMH decreased similarly in both groups during the treatment, reassuring safety of developing seminiferous epithelium in both treatment approaches. Since a fixed dose of Lz induced variable serum Lz levels with a desired puberty-promoting effect in all boys, more research is needed to aim at a minimal efficient dose per weight. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Academy of Finland, the Foundation for Pediatric Research, the Emil Aaltonen Foundation, Sigrid Juselius Foundation and Helsinki University Hospital Research Funds. The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: NCT01797718.

Disorders of sex development: timing of diagnosis and management in a single large tertiary center.

BACKGROUND: We describe the phenotypic spectrum and timing of diagnosis and management in a large series of patients with disorders of sexual development (DSD) treated in a single pediatric tertiary center. METHODS: DSD patients who had visited our tertiary center during the survey period (between 2004 and 2014) were identified based on an ICD-10 inquiry, and their phenotypic and molecular genetic findings were recorded from patient charts. RESULTS: Among the 550 DSD patients, 53.3% had 46,XY DSD; 37.1% had sex chromosome DSD and 9.6% had 46,XX DSD. The most common diagnoses were Turner syndrome (19.8%, diagnosed at the mean age of 4.7 ± 5.5 years), Klinefelter syndrome (14.5%, 6.8 ± 6.2 years) and bilateral cryptorchidism (23.1%). Very few patients with 46,XY DSD (7%) or 46,XX DSD (21%) had molecular genetic diagnosis. The yearly rate of DSD diagnoses remained stable over the survey period. After the release of the Nordic consensus on the management of undescended testes, the age at surgery for bilateral cryptorchidism declined significantly (P < 0.001). CONCLUSIONS: Our results show that (i) Turner syndrome and Klinefelter syndrome, the most frequent single DSD diagnoses, are still diagnosed relatively late; (ii) a temporal shift was observed in the management of bilateral cryptorchidism, which may favorably influence patients' adulthood semen quality and (iii) next-generation sequencing methods are not fully employed in the diagnostics of DSD patients.

Prevalence and characteristics of diabetes among Somali children and adolescents living in Helsinki, Finland.

OBJECTIVE: We compared the prevalence and characteristics of diabetes between Somali and Finnish children in the City of Helsinki. SUBJECTS AND METHODS: Ten Somali and 310 non-Somali children <16 yr of age were treated for diabetes in Helsinki at the end of 2007. We analyzed autoantibodies, HLA alleles, and serum 25-hydroxy-vitamin D [S25(OH)D] concentrations. RESULTS: The prevalence of diabetes was 40/10,000 (95% CI 19-73/10,000) for the Somali children and 37/10,000 (95% CI 33-41/10,000) for the background population. At least one autoantibody was detected in all seven Somali patients sampled within 18 months after the diagnosis. Most Somalis (75%) carried HLA-conferred susceptibility to type 1 diabetes (T1D), DR3-DQ2 being the dominating HLA haplotype. Low S25(OH)D levels (<40 nmol/L) were seen in 83% of the Somali patients and in 60% of their siblings. CONCLUSIONS: These data show that (i) Somali children have autoimmune diabetes, (ii) the prevalence of T1D is similar among Somali and Finnish children, and (iii) both affected and unaffected Somali children have low concentrations of S25(OH)D.

Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation.

AIMS/HYPOTHESIS: Epidermal growth factor receptor (EGFR) signalling is essential for the proper fetal development of pancreatic islets and in the postnatal formation of an adequate beta cell mass. In this study we investigated the role of EGFR signalling in the physiological states of beta cell mass expansion in adults during metabolic syndrome and pregnancy, as well as in regeneration after pancreatic duct ligation. METHODS: Heterozygous Pdx1-EGFR-dominant-negative (E1-DN) mice, which have a kinase-negative EGFR under the Pdx1 promoter, and wild-type mice were both subjected to a high-fat diet, pregnancy and pancreatic duct ligation. RESULTS: The beta cell mass of wild-type mice fed the high-fat diet increased by 70% and the mice remained normoglycaemic; the E1-DN mice became diabetic and failed to show any compensatory beta cell mass expansion. Similarly, pregnant wild-type mice had four times more proliferating beta cells and a 75% increase in beta cell mass at mid-gestation, in contrast to the pregnant E1-DN mice, which did not show any significant beta cell compensation and were hyperglycaemic in an intraperitoneal glucose tolerance test. However, after pancreatic duct ligation, both the wild-type and E1-DN mice showed similar expression of Ngn3 (also known as Neurog3) and beta cell proliferation increased to a similar level in the ligated part of pancreas. CONCLUSIONS/INTERPRETATIONS: EGFR signalling is essential in beta cell mass expansion during a high-fat diet and pregnancy where replication is the primary mechanism for compensatory beta cell mass expansion. In contrast, EGFR signalling appears not to be crucial to increased beta cell proliferation after pancreatic duct ligation.

ERBB receptors in developing, dysplastic and malignant oral epithelia.

Some oral squamous cell carcinomas (OSCCs) overexpress epidermal growth factor receptor (EGFR) but little is known about the receptor system overall during oral carcinogenesis. We studied all four ERBB receptors (EGFR, ERBB2-4) in developing (n=2), normal (n=7), dysplastic (n=23) and malignant (n=26) oral epithelia by means of immunohistochemistry. The investigations were supplemented by conducting reverse transcription-polymerase chain reactions in relation to 13 OSCC samples. All four ERBB receptors were detected in developing oral epithelium and, to a lesser degree, in mature oral epithelium. An increase in EGFR immunoreactivity was seen in 61% and 54% of dysplasias and OSCCs, respectively. The corresponding percentages for ERBB2 were 48 and 12, for ERBB3 48 and 43. ERBB4 nuclear staining was increased in 30% of dysplasias and 26% of OSCCs. Changes in ERBB receptor mRNA levels were not statistically significant. The results show that ERBB receptor profiles are specific to each tumour. Increased nuclear translocation of ERBB4 in some OSCCs may alter transcription of target genes and be associated with cancer progression. This information may be useful for clinicians as EGFR inhibitors are becoming treatment options in modern oncology.

Novel splice site CACNA1A mutation causing episodic ataxia type 2.

Episodic ataxia type 2 (EA-2) is an autosomal dominant neurological disorder, characterized by episodes of ataxia, vertigo, nausea, nystagmus, and fatigue, associated with acetazolamide responsiveness. The disease is caused by mutations in the P/Q-type calcium channel Ca(v)2.1 subunit gene, CACNA1A, located on chromosome 19p13.2. We analyzed a family with 13 affected individuals for linkage to this locus and reached a two-point maximum LOD score of 4.48. A novel CACNA1A mutation, IVS36-2A>G, at the 3' acceptor splice site of intron 36 was identified by sequencing. It is the first described CACNA1A acceptor splice site mutation and the most C-terminal EA-2-causing mutation reported to date.

Different mechanisms of syndecan-1 activation through a fibroblast-growth-factor-inducible response element (FiRE) in mucosal and cutaneous wounds.

Syndecan-1 expression is enhanced in cutaneous and mucosal wounds. We have previously demonstrated that wounding-induced syndecan-1 expression in the skin occurs transcriptionally, through a fibroblast-growth-factor-inducible element (FiRE). Here, we show that FiRE is also activated in mucosal wounds. However, both the expression patterns and the activation mechanisms of FiRE are different from those in the skin. In the mucosa in vivo, the activation starts and ends earlier than in cutaneous wounds. FiRE is first detected at around 12 hours in keratinocytes, and the activation declines by the third day after wounding occurs. The activation is seen on the migrating sheet of epithelial mucosa, as in the case of cutaneous wounding. In contrast to the situation in vivo, organ-cultured mucosal wounds exhibit no FiRE activity, while organ-cultured cutaneous wounds show robust activity. Activation in mucosal wounds is enhanced, however, by the application of epidermal growth factor. This suggests that exogenous growth factor activity is required for activation of syndecan-1 in mucosal wounds but not in cutaneous wounds.

Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors.

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various growth factors. Epidermal growth factor receptor (EGF-R) is expressed throughout the developing pancreas. We have analyzed here the pancreatic phenotype of EGF-R deficient (-/-) mice, which generally die from epithelial immaturity within the first postnatal week. The pancreata appeared macroscopically normal. The most striking feature of the EGF-R (-/-) islets was that instead of forming circular clusters, the islet cells were mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal EGF-R (-/-) beta-cells was significantly reduced (2.6+/-0.4 versus 5.8+/-0.9%, P<0.01) and the difference persisted even at 7-11 days of age. Analysis of embryonic pancreata revealed impaired branching morphogenesis and delayed islet cell differentiation in the EGF-R (-/-) mice. Islet development was analyzed further in organ cultures of E12.5 pancreata. The proportion of insulin-positive cells was significantly lower in the EGF-R (-/-) explants (27+/-6 versus 48+/-8%, P<0.01), indicating delayed differentiation of the beta cells. Branching of the epithelium into ducts was also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity was reduced 20% in EGF-R (-/-) late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-1 (PAI-1) were markedly higher, while no apparent differences were seen in the levels of active uPA and tPa between EGF-R (-/-) and wild-type pancreata. Our findings suggest that the perturbation of EGF-R-mediated signalling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-1 production and decreased gelatinolytic activity correlated to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development.

Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure.

Craniofacial malformations are among the most frequent congenital birth defects in humans; cleft palate, that is inadequate fusion of the palatal shelves, occurs with an annual incidence of 1 in 700 to 1 in 1,000 live births among individuals of European descent. The secondary palate arises as bilateral outgrowths from the maxillary processes, and its formation depends on the coordinated development of craniofacial structures including the Meckel's cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-alpha (TGF-alpha), an epidermal growth factor receptor (EGFR) ligand made by most epithelia. Here we have characterized craniofacial development in Egfr-deficient (Egfr-/-) mice. Newborn Egfr-/- mice have facial mediolateral defects including narrow, elongated snouts, underdeveloped lower jaw and a high incidence of cleft palate. Palatal shelf explants from Egfr-/- mice fused, but frequently had residual epithelium in the midline. In addition, morphogenesis of Meckel's cartilage was deficient in cultured mandibular processes from Egfr-/- embryos. The secretion of matrix metalloproteinases (MMPs) was diminished in Egfr-/- explants, consistent with the ability of EGF to increase MMP secretion and with the decreased MMP expression caused by inhibition of Egfr signalling in wild-type explants. Accordingly, inactivation of MMPs in wild-type explants phenocopied the defective morphology of Meckel's cartilage seen in Egfr-/- explants. Our results indicate that EGFR signalling is necessary for normal craniofacial development and that its role is mediated in part by its downstream targets, the MMPs, and may explain the genetic correlation of human cleft palate with polymorphisms in TGFA.

Uterine and vaginal organ growth requires epidermal growth factor receptor signaling from stroma.

Estrogens are crucial for growth and function of the female genital tract. Recently, we showed that induction of uterine epithelial proliferation by estradiol is a paracrine event requiring an estrogen receptor-positive stroma. Growth factors [such as EGF (epidermal growth factor) ligands] are likely paracrine mediators, which may directly or indirectly regulate epithelial proliferation in estrogen target organs via cell-cell interactions. In this report, we used mice with a null mutation in their EGF receptor (EGFR) to examine the role of EGFR signaling in growth of the uterus and vagina and in estrogen-induced uterine and vaginal epithelial proliferation. When WT and EGFR-knockout (EGFR-KO) uteri and vaginae were grown as renal capsule grafts in nude mice, growth of uterine and vaginal grafts of EGFR-KO mice was reduced, compared with their WT counterparts. Grafts of both EGFR-KO uteri and vaginae were about one third smaller (wet weight) than their corresponding WT organs, even though differentiation of both epithelium and mesenchyme were normal in both cases. Both wild-type and EGFR-KO vaginal grafts contained within their lumina alternating layers of cornified and mucified epithelial cell layers, indicating cyclic alteration of epithelial differentiation. In response to estradiol treatment, stromal cell labeling index (LI), as assessed by incorporation of 3H-thymidine, was severely depressed in EGFR-KO uterine and vaginal grafts vs. stromal cell LI in WT uterine and vaginal grafts. Unexpectedly, epithelium of both EGFR-KO and wild-type grafts responded comparably to estradiol with a marked elevation (approximately 7-fold overall) of epithelial LI in response to estradiol in uterine and vaginal epithelia. These data supported the hypothesis that overall uterine and vaginal organ growth, in response to estrogen, required EGFR signaling for DNA synthesis in the fibromuscular stroma, whereas EGFR signaling was not essential for estrogen-induced epithelial growth in the uterus and vagina.

Epidermal growth factor receptor as a mediator of developmental toxicity of dioxin in mouse embryonic teeth.

We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralization defects in the developing teeth of their children. Developmental dental defects have also been reported in rhesus macaques and rats experimentally exposed to dioxin. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in dental toxicity, we cultured embryonic molar teeth from EGFR-deficient mice with TCDD, epidermal growth factor (EGF), and both agents in combination. In teeth of the normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization, the enamel matrix was not deposited, and cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR-deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of the normal embryos. These results show that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus, EGFR may also play a role in the developmental defects that dioxins cause in human teeth. Because EGFR is widely expressed in developing organs, EGFR signaling may even be of general relevance in the mediation of the developmental toxicity of TCDD.

Impaired lung branching morphogenesis in the absence of functional EGF receptor.

The mammalian lung develops through branching morphogenesis which is controlled by growth factors, hormones, and extracellular matrix proteins. We have evaluated the role of EGF-receptor signaling in lung morphogenesis by analyzing the developmental phenotype of lungs in mice with an inactivated the EGF-receptor gene both in vivo and in organ culture. Neonatal EGF-receptor-deficient mice often show evidence of lung immaturity which can result in visible respiratory distress. The lungs of these mutant mice had impaired branching and deficient alveolization and septation, resulting in a 50% reduction in alveolar volume and, thus, a markedly reduced surface for gas exchange. The EGF-receptor inactivation also resulted in type II pneumocyte immaturity, which was apparent from their increased glycogen content and a reduced number of lamellar bodies. The defective branching was already evident at Day 12 of embryonic development. When explants of embryonic lungs from Day 12 embryos were cultured under defined conditions, the branching defect in EGF-receptor-deficient lungs was even more pronounced, with only half as many terminal buds as normal lungs. EGF treatment stimulated the expression of surfactant protein C and thyroid transcription factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor signaling regulates the expression of these marker genes during type II pneumocyte maturation. Taken together, our data indicate that signal transduction through the EGF receptor plays a major role in lung development and that its inactivation leads to a respiratory distress-like syndrome.

Epidermal growth factor receptor in mice and men--any applications to clinical practice?

The epidermal growth factor receptor (EGF-R) is perhaps the best studied member of tyrosine kinase receptors. Its inactivation by homologous recombination results in three different phenotypes ranging from peri-implantation lethality to postnatal lethality. The mildest form of EGF-R inactivation leads to epithelial immaturity and postnatal death due to respiratory failure and necrotizing enterocolitis-like lesions in the intestine. The defects seen in this 'postnatal lethality phenotype' manifest in the classical EGF-responsive organs (skin, intestine) and organs undergoing branching morphogenesis during development (lung, kidney, mammary gland, pancreas and prostate), and thus accord with the concept of EGF family members being important epithelial mitogens. The respiratory failure of the EGF-R (-/-) mice results from impaired branching of the alveolar tree and leads to decreased surface for gas exchange. Overall, the lung phenotype bears similarity to respiratory distress syndrome and bronchopulmonary dysplasia--the most common complications of prematurity in humans. Intestinal changes seen in the EGF-R (-/-) mice vary in severity, the end-point being severe mucosal lesions and necroses. These findings resemble those seen in necrotizing enterocolitis of premature babies, a serious intestinal problem in the neonate. Although deficient EGF-R function is not the reason for these prematurity-associated diseases it may nevertheless exacerbate them. Potential usage of EGF transforming growth factor-alpha in clinical work is discussed.

A WD-domain protein that is associated with and phosphorylated by the type II TGF-beta receptor.

Transforming growth factor-beta (TGF-beta) is the prototype for a family of extracellular polypeptides that affect cell proliferation and differentiation, and tissue morphogenesis. TGF-beta signalling is mediated by two types of serine/threonine kinase receptors, the type I and II receptors, which are able to form a heteromeric complex. No cytoplasmic proteins that associate with these receptors in vivo, or are their kinase targets, have yet been described. We have now identified a WD-domain-containing protein, TRIP-1, which specifically associates with the type II TGF-beta receptor in a kinase-dependent way. TRIP-1 does not interact with the type II activin or type I receptors, but associates with the heteromeric TGF-beta receptor complex. TRIP-1 is phosphorylated on serine and threonine by the receptor kinase, strongly suggesting that it has a role in TGF-beta signalling. This is supported by coexpression of TRIP-1 and type II receptor during development. The existence of TRIP-1 homologues in plant and yeast suggests a conserved function in all eukaryotes.

Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.

Since the discovery that epidermal growth factor (EGF) can accelerate opening of the eyelids, the EGF receptor (EGF-R) has been extensively studied and is now considered to be a prototype tyrosine kinase receptor. Binding of EGF or of transforming growth factor-alpha (TGF-alpha) or other related factors activates the receptor and induces cell proliferation and differentiation. Although it is not found on haematopoietic cells, the EGF-R is widely expressed in mammals and has been implicated in various stages of embryonic development. Here we investigate the developmental and physiological roles of this receptor and its ligands by inactivating the gene encoding EGF-R. We find that EGF-R-/- mice survive for up to 8 days after birth and suffer from impaired epithelial development in several organs, including skin, lung and gastrointestinal tract.

TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors.

The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.

Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas.

An increasing body of evidence suggests that insulin-like growth factors (IGFs) are important in the development of some tumors. In the present study, we investigated the gene expression of IGF-I, IGF-II, and their receptors in different adrenal tumors and hyperplasias. Four adrenocortical carcinomas, 15 adenomas, 4 pheochromocytomas, 5 nodular hyperplasias, and hyperplastic adrenals from 2 patients with pituitary Cushing's disease were analyzed and compared to normal adrenals. Northern blots, dot blots, and reverse transcription polymerase chain reaction analyses were used for mRNA detection, and immunohistochemistry was used for IGF-II peptide localization. The IGF-I mRNA content was low in normal, hyperplastic, and neoplastic adrenals. IGF-II mRNA levels were more than 10-fold higher in hormonally active adrenocortical carcinomas than in normal adult adrenals, and increased IGF-II-like immunoreactivity was detectable in these carcinomas. A moderate elevation of the IGF-II mRNA content was also noted in one nonfunctioning carcinoma. The IGF-II mRNA content was high in pheochromocytomas, as reported previously. Furthermore, Cushing's and Conn's adenomas expressed IGF-II mRNA levels similar to those in the normal adrenal. In nodular adrenocortical hyperplasia, we found variable IGF-II mRNA content (40-400% of normal adrenal expression). All of the normal adrenal and tumor tissues studied contained both the type I and type II IGF receptor mRNAs. The findings in the present study show that both IGF-I and IGF-II and their receptor mRNAs are expressed in various adrenocortical tumors. Moreover, the high IGF-II expression in functional adrenocortical carcinomas suggests that the IGFs may be involved in the auto/paracrine regulation of certain adrenocortical tumors.

EGF receptor and its ligands, EGF and TGF-alpha, in developing and neoplastic human odontogenic tissues.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate cell proliferation and functional maturation through the EGF receptor (EGF-R). Their roles in human tooth development and odontogenic tumorigenesis have not been explored. We studied the expression of EGF, TGF-alpha and EGF-R in human fetal teeth (cap stage to early hard tissue formation) and various odontogenic tumors. EGF-R mRNA and immunoreactive cells were mostly located in odontogenic epithelium. EGF-R expression was subject to temporospatial variation at different stages of tooth development. EGF and TGF-alpha mRNAs were detected in fetal teeth only by the reverse transcription polymerase chain reaction (RT-PCR). However, EGF and TGF-alpha immunoreactive cells were demonstrated in epithelial elements of tooth germ, suggesting that the peptides partially originate from non-odontogenic sources. In odontogenic tumors, EGF-R mRNA and immunoreactivity were confined to neoplastic epithelium. Transcripts for TGF-alpha but not for EGF were detected in tumors of odontogenic epithelial, epithelial-ectomesenchymal and ectomesenchymal origins. It is concluded that regulation of EGF-R expression is developmentally regulated in human odontogenesis. Furthermore, the odontogenic epithelium is the main target tissue for both EGF and TGF-alpha during tooth development. TGF-alpha and its receptor may also be involved in odontogenic tumorigenesis.

Insulin-like growth factor-II and transforming growth factor-alpha in developing human fetal pancreatic islets.

To understand the development of the human pancreas better, we studied the expression and regulation of insulin, insulin-like growth factor-II (IGF-II) and transforming growth factor-alpha (TGF-alpha) genes in the human fetal pancreas and islet-like cell clusters (ICC) from the second trimester human fetuses. Northern blot analysis revealed an abundant expression of IGF-II, insulin and TGF-alpha mRNAs in the intact pancreas and the cultured ICCs. Furthermore, transcripts for insulin receptor, type-1 and -2 IGF receptors, and GH receptor could be amplified by polymerase chain reaction analysis from the pancreas and the ICCs. With in-situ hybridization, IGF-II mRNA was found in abundance in both the exocrine and endocrine pancreas, exceeding the amount of insulin mRNA. In ICCs, insulin mRNA-containing cells were present as small clusters in the periphery and in the centre of the clusters corresponding to the immunolocation of insulin. The ICCs also contained many epidermal growth factor-, insulin- and type-1 IGF receptor- and TGF-alpha-positive cells. When the ICCs were cultured in the presence of various secretagogues, only dibutyryl cyclic AMP was found to up-regulate insulin mRNA (39%; P < 0.05). IGF-II mRNA was also under cyclic AMP-dependent regulation (threefold increase; P = 0.025). Furthermore, blocking the type-1 IGF receptor with a monoclonal receptor antibody drastically reduced insulin expression (87%; P = 0.005) and additionally down-regulated IGF-II mRNA (49%; P = 0.005). IGF-1, IGF-II, TGF-alpha or epidermal growth factor-receptor antibody had no significant effect on either insulin or IGF-II mRNA. Exogenous TGF-alpha inhibited the release of insulin by the ICCs. It was concluded that IGF-II and TGF-alpha may be involved in the regulation of islet growth and differentiation.