Water deprivation stimulates transforming growth factor-beta 2 accumulation in the juxtaglomerular apparatus of mouse kidney.

Transforming growth factor-beta (TGF-beta) modulates the growth and differentiation of many cells and often functions in an autocrine or paracrine fashion. The myoepithelial cells of the renal juxtaglomerular apparatus (JGA) synthesize and secrete renin. Under conditions which chronically stimulate renin production, the JGA undergoes hypertrophy and hyperplasia. The molecular factors responsible for these changes in the JGA have not been identified. In the present study, plasma renin activity was stimulated in the mouse by water deprivation. Using immunoperoxidase staining with specific antibodies against TGF-beta 1, beta 2, and beta 3, we found increased TGF-beta 2 accumulation in the JGA and interlobular arteries. Immunostaining with renin antiserum demonstrated colocalization of TGF-beta 2 and renin. TGF-beta 1 and beta 3 expression was not different between control and water-deprived mice. Our results suggest that in the setting of water deprivation, TGF-beta 2 is localized in a manner which would allow it to act either as a growth factor for or as a phenotypic modulator of the JGA and renal arterioles.

Association of transforming growth factor-beta 1 with prostate cancer: an immunohistochemical study.

Prostate tissue samples from patients with prostatic carcinoma (PC) and/or benign prostatic hyperplasia (BPH) were examined for expression of transforming growth factor-beta 1 (TGF-beta 1) using an immunohistochemical technique. Tissues were stained with CC and LC antisera, which react with extracellular and intracellular TGF-beta 1, respectively. All PC and BPH tissues showed positive extracellular staining; however, CC-immunoreactive material was significantly more extensive in PC compared with BPH, the average positively staining areas being 59% and 26%, respectively. This differential staining pattern was evident in cases in which areas of PC were located adjacent to areas of BPH. LC staining was identified exclusively intracellularly involving both stromal and epithelial cells in cases of PC as well as BPH. However, while stromal cell staining was more pronounced in BPH, epithelial cell staining tended to be more extensive and more intense in PC. The findings suggest that TGF-beta 1 may be biologically important in the development of PC and BPH.

Localization of transforming growth factor-beta isotypes in lesions of the human breast.

Transforming growth factor-beta s (TGF-beta) comprise a highly conserved family of multifunctional cell regulatory peptides that may play a role in a variety of pathologic processes. To date, five TGF-beta isotypes have been identified, three of these in mammalian systems. A number of cultured human breast carcinoma cell lines produce biologically inactive latent TGF-beta and are growth inhibited by activated TGF-beta; TGF-beta production is estrogen-influenced in some of these cell lines. To investigate the potential role of the TGF-beta isotypes in human breast disease, we localized TGF-beta s 1, 2, and 3 immunohistochemically in normal breast, fibrocystic change, epithelial hyperplasia, sclerosing adenosis, fibroadenoma, cystosarcoma phyllodes, and several carcinoma variants. Transforming growth factor-beta s 1, 2, and 3 were identified intracellularly in most active mammary epithelia, regardless of the lesion, including carcinoma; the associated stroma contained little or no intracellular TGF-beta. An antibody that recognizes an extracellular conformation of TGF-beta stained normal intralobular stroma and, more extensively, the stroma of active fibroadenomas and low-grade phyllodes tumors and the desmoplastic stroma of carcinomas. The results indicate the potential for paracrine and autocrine regulation of the mammary gland by TGF-beta and suggest an association between TGF-beta and abnormal stromal proliferations. Altered expression of TGF-beta s 1, 2, and 3 at the protein level in mammary epithelia appears not to be a major feature of most breast lesions, raising the possibility that altered cellular response to the TGF-beta already present may play a role in the development of breast disease.

Argon laser therapy of vulvar angiokeratoma.

A 25-year-old woman with Noonan syndrome presented with painful, bleeding lesions of the vulva and perineum. The histologic diagnosis was angiokeratoma. Chronic increased central venous pressure from the patient's underlying cardiac disease was felt to be etiologic. The Argon laser was used for ablation, with minimal blood loss despite the extreme vascularity of these lesions.

Elevated second trimester maternal serum alpha-fetoprotein and cytomegalovirus infection.

A case is presented of intrauterine cytomegalovirus infection. Initially, the infection was most probably manifested by elevated second trimester maternal serum alpha-fetoprotein levels. Placental involvement by the infectious agent may have produced the elevations in maternal serum. Alpha-fetoprotein elevation unrelated to fetal anatomic abnormalities, twins, or incorrect dates may represent a potential marker of placental pathology.

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location.

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.

Presence of a major (storage?) protein in dormant spores of the fungus Botryodiplodia theobromae.

Approximately 23% of the protein isolated from dormant spores of Botryodiplodia theobromae consisted of a single polypeptide; the polypeptide is probably degraded during germination.

Immunohistological demonstration of transforming growth factor-beta isoforms in the skin of patients with systemic sclerosis.

We investigated the expression of transforming growth factor-beta (TGF-beta) isoforms in involved and uninvolved areas of skin from patients with systemic sclerosis (SSc) and normal controls. Paraffin-embedded skin specimens were stained for TGF-beta 1, TGF-beta 2, and TGF-beta 3 using the avidin-biotin-peroxidase technique. TGF-beta 2 was expressed intensely in the extracellular matrix of the skin biopsies obtained from involved areas of patients with SSc, in contrast to the uninvolved areas and normal individuals. TGF-beta 2 was deposited throughout the entire dermis and also in a linear fashion along the dermoepidermal junction and in the perivascular areas of SSc-involved skin. Although infiltrating mononuclear cells were not present in great numbers, they did not stain for TGF-beta 2. TGF-beta 1 and TGF-beta 3 were not expressed in the extracellular space in either patients or normal controls, except in rare cases. The cellular staining which was observed for all three isoforms did not differ between involved and uninvolved skin and normal controls. The finding of increased deposition of TGF-beta 2 in the involved skin of patients with SSc implies that it may be involved in the pathologic fibrotic process.

Expression of transforming growth factor-beta isoforms in small round cell tumors of childhood. An immunohistochemical study.

The transforming growth factor (TGF)-betas are a highly conserved group of potent multifunctional cell regulatory proteins with variable effects on cell growth and differentiation. Most of the small round cell group of childhood tumors are thought to arise from either primitive mesenchyme or neuroectoderm and show evidence of skeletal muscle or neural differentiation, and rarely both. To investigate the possibility that the TGF-betas have a role in the growth or differentiation of these neoplasms, we used antibodies specific for peptide sequences of the three known mammalian TGF-beta isoforms (TGF-betas 1, 2, and 3) to probe for TGF-beta protein expression in a total of 49 cases. TGF-beta 1 immunoreactivity was present in 16/17 (94%) of rhabdomyosarcomas, and the staining intensity was usually strong. TGF-beta 1 was also present in three of three ectomesenchymomas. In contrast, TGF-beta 1 was absent in all but one out of nine poorly differentiated neuroblastomas. Differentiating neuronal cells of ganglioneuroblastomas, however, were strongly positive for TGF-beta 1. Ewing's sarcomas and peripheral primitive neuroectodermal tumors had a less consistent, but usually positive, staining pattern. TGF-beta 3 staining patterns were very similar to those of TGF-beta 1. TGF-beta 2 immunoreactivity was only rarely detected in this group of tumors. The results suggest different roles for TGF-betas 1 and 3 in neuroblastoma and rhabdomyosarcoma. Expression of TGF-betas 1 and 3 is associated with neuronal differentiation of neuroblastoma. In contrast, these proteins may promote the growth of rhabdomyosarcoma by suppressing differentiation.

Renal vascular induction of TGF-beta 2 and renin by potassium depletion.

Recently, we have found that transforming growth factor (TGF)-beta 2 and renin are abundantly expressed in the juxtaglomerular apparatus (JGA) of dehydrated mice. Since potassium (K+) depletion also stimulates renin and induces hypertrophy of the JGA, we examined the ability of this maneuver to stimulate TGF-beta isoforms and renin in renovascular tissue and the JGA of young rats. Sprague-Dawley rats (50 +/- 5 g) were fed either a control diet or a potassium-deficient diet (< 0.05% K+) for 7, 16, or 21 days. As a control for TGF-beta and renin stimulation, an additional group of animals was fed a normal diet but was water deprived for three days. Potassium-depleted animals experienced severe growth retardation but kidney weight increased significantly. Potassium depletion induced both TGF-beta 2 and renin immunoreactivity in renal arterioles and the JGA but had no effect on TGF-beta 1 and TGF-beta 3 isoforms. To determine the role of circulating angiotensin II in the stimulation of TGF-beta 2 by potassium depletion, a group of potassium-depleted rats received enalapril (100 mg/liter) in the drinking water. The addition of converting enzyme inhibitor increased both the intensity of TGF-beta 2 and renin staining as well as the number of cells positively stained. Our results demonstrate that K+ depletion induces TGF-beta 2 and renin in renal arterioles and in the JGA. Furthermore, circulating angiotensin II is not responsible for the increase in the local expression of TGF-beta 2. These findings suggest that TGF-beta 2 may be an important mediator of JGA hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat.

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.

Transgenic models for the study of prostate cancer.

Transgenic model systems provide tools for obtaining information that clarifies important relationships between genetic alterations and carcinogenesis. One such relationship is the induction of specific growth factor activities by dominantly acting oncogenes. Using a "transgenic organ" model referred to as mouse prostate reconstitution (MPR) under conditions where the ras and myc oncogenes were introduced using a recombinant retrovirus into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer (PC) was produced with high frequency (> 90%) in inbred C57BL/6 mice. Time-course studies using northern blotting and immunohistochemical analysis showed that the transition from benign to malignant status invariably was associated with the induction of elevated transforming growth factor-beta 1 (TGF-beta 1) expression. Additional immunohistochemical analysis of TGF-beta 1 in human PC and benign prostatic hyperplasia (BPH) showed that positive extracellular staining was significantly more extensive in PC compared with BPH. This differential staining pattern was evident in focal areas of PC adjacent to BPH. These findings in both the MPR model system and human PC suggest that elevated TGF-beta 1 expression is involved in the progression to malignancy and that its pattern of expression may become a useful marker of PC. Additional studies using transgenic animal models will continue to provide important clinically useful information about PC in man.

The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction.

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.

Transforming growth factor beta 1 as a biomarker for prostate cancer.

Using the mouse prostate reconstitution (MPR) model system, under conditions where the ras and myc oncogenes are introduced via a recombinant retrovirus into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer is produced with high frequency (> 90%) using inbred C57BL/6 mice. Northern blotting and immunohistochemical analysis showed that the transition from benign prostatic hyperplasia (BPH) to prostate cancer is invariably associated with the induction of elevated transforming growth factor-beta 1 (TGF-beta 1) expression. Similar analysis of TGF-beta 1 in human BPH and prostate cancer is consistent with our MPR results and indicates that the accumulation of extracellular TGF-beta 1 is significantly more intense in prostate cancer compared to normal or benign prostate tissues. Interestingly, where benign pathologies are observed in the prostatic stroma in the presence of benign prostatic epithelium, extracellular TGF-beta 1 is seen predominantly in the stromal compartment. Experimental studies clearly demonstrate that mRNA levels of TGF-beta 1 and other growth related genes are regulated by androgens in prostate cancer cells. Overall, our results suggest that elevated TGF-beta 1 is involved in the development of prostate cancer. Direct determination of TGF-beta 1 levels and distribution as well as analysis of localized and systemic effects produced by TGF-beta 1 may serve as useful biomarkers for prostate cancer.

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways.

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.

Roles of trk family neurotrophin receptors in medullary thyroid carcinoma development and progression.

Although initiating mutations in the ret protooncogene have been found in familial and sporadic medullary thyroid carcinoma (MTC), the molecular events underlying subsequent tumor progression stages are unknown. We now report that changes in trk family neurotrophin receptor expression appear to be involved in both preneoplastic thyroid C cell hyperplasia and later tumor progression. Only a subset of normal C cells expresses trk family receptors, but, in C cell hyperplasia, the affected cells consistently express trkB, with variable expression of trkA and trkC. In later stages of gross MTC tumors, trkB expression was substantially reduced, while trkC expression was increased and often intense. In a cell culture model of MTC, exogenous trkB expression resulted in severely impaired tumorigenicity and was associated with 11-fold lower levels of the angiogenesis factor vascular endothelial growth factor. These results suggest that trk family receptor genes participate in MTC development and progression, and, in particular, that trkB may limit MTC tumor growth by inhibition of angiogenesis.