Faculty Bibliography

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus

Tamoxifen has long been the endocrine treatment of choice for women with breast cancer and is now employed for prophylactic use in women at high risk from breast cancer. Other selective estrogen receptor modulators (SERMs), such as raloxifene, mimic some of tamoxifen's beneficial effects and, like tamoxifen, exhibit a complex mixture of organ-specific estrogen agonist and antagonistic properties. However, accompanying the positive effects of tamoxifen has been the emergence of evidence for an increased risk of endometrial cancer associated with its use. A more complete understanding of the mechanism(s) of SERM carcinogenicity and endometrial effects is therefore required. We have sought to compare and characterise the transcript profile of tamoxifen, raloxifene and the agonist estradiol in human endometrial cells. Using primary cultures of human endometria, to best emulate the in vivo responses in a manageable in vitro system, we have shown 230 significant changes in gene expression for epithelial cultures and 83 in stromal cultures, either specific to 17beta-estradiol, tamoxifen or raloxifene, or changed across more than one of the treatments. Considering the transcriptome as a whole, the endometrial responses to raloxifene or tamoxifen were more similar than either drug was to 17beta-estradiol. Treatment of endometrial cultures with tamoxifen resulted in the largest number of gene changes relative to control cultures and a high proportion of genes associated with regulation of gene transcription, cell-cycle control and signal transduction. Tamoxifen-specific changes that might point towards mechanisms for its proliferative response in the endometrium included changes in retinoblastoma and c-myc binding proteins, the APCL, dihydrofolate reductase (DHFR) and E2F1 genes and other transcription factors. Tamoxifen was also found to give rise to the highest number of gene expression changes common to those that characterise malignant endometria. It is anticipated that this study will provide leads for further and more focused investigation into SERM carcinogenicity

Transforming growth factor beta (TGF-beta), a potent ubiquitous endogenous inhibitor of epithelial cell growth, is secreted as a latent molecule (LTGF-beta)requiring activation for function. TGF-beta signals through the type I(TbetaRI) and type II (TbetaRII) receptors, which cooperate to phosphorylate/activate Smad2/3, the transcriptional regulators of genes that induce cell cycle arrest. That carcinomas grow in vivo suggests that they are refractory to TGF-beta. However, this has been difficult to prove because of an inability to analyze the functional status of TGF-beta in vivo as well as lack of close physiological paradigms for carcinoma cells in vitro. The current studies demonstrate that whereas primary cultures of endometrial epithelial cells derived from normal proliferative endometrium (PE; n = 10) were dose-dependently and maximally growth inhibited by 55% +/- 5.3% with 10 pM TGF-beta1, endometrial epithelial cells derived from endometrial carcinomas (ECAs; n = 10) were unresponsive (P < or = 0.0066). To determine the mechanism of TGF-beta resistance in ECAs, we analyzed the TGF-beta signaling pathway in vivo by immunohistochemistry using specific antibodies to TbetaRI and TbetaRII, Smads, and to the phosphorylated form of Smad2 (Smad2P), an indicator of cells responding to bioactive TGF-beta. Smad2P was expressed in all of the normal endometria (n = 25), and was localized to the cytoplasm and nucleus in PE, and only nuclear in the secretory endometrium. In marked contrast, Smad2P immunostaining was weak or undetectable in ECA (n = 22; P < or = 0.001) and reduced in glandular hyperplasia (n = 25) compared with normal endometrium. However, total Smad2 and Smad7 (which inhibits Smad2 activation) levels were identical in ECA and normal tissue. Consistent with loss of downstream signaling, both TbetaRI (n = 19) and TbetaRII (n = 22) protein expression were significantly reduced in ECA compared with PE (n = 11; P < or = 0.05). By in situ hybridization, the mRNA levels of TbetaRI and TbetaRII were decreased in the carcinoma cells compared with normal PE glands, suggesting that receptor down-regulation occurs at the transcriptional level. Furthermore, a somatic frameshift mutation in the polyadenine tract at the 5' end of the TbetaR-II gene was detected in two of six cases examined. Finally, the ability of explants of ECA to activate endogenous LTGF-beta was deficient compared with normal tissue (23.5% versus 7.4%). Therefore, our results suggest that loss of Smad2 signaling in ECA may be because of down-regulation of TbetaRI and TbetaRII, and/or decreased activation of LTGF-beta. Because disruption of TGF-beta signaling occurred independent of grade or degree of invasion and was evident in premalignant hyperplasia, we conclude that inactivation of TGF-beta signaling leading to escape from normal growth control occurs at an early stage in endometrial carcinogenesis, thereby defining novel molecular targets for cancer prevention

In a rat model of lung injury induced by the antineoplastic antibiotic, bleomycin, there is loss of type I alveolar epithelial cells (AECs) followed by infiltration of activated inflammatory cells, type II AEC proliferation, and fibrosis. At 4 and 7 days after bleomycin administration alveolar macrophages have increased production and release of active transforming growth factor (TGF)-beta1, an inhibitor of epithelial cell proliferation. Paradoxically at these same time intervals there is a concomitant induction of type II AEC proliferation. For TGF-beta-mediated signal transduction to occur, the expression of both TCF-beta receptor types I (TbetaR-I) and II (TbetaR-II) must be present. Using immunohistochemistry and in situ hybridization, 4 and 7 days after bleomycin administration the expression of TbetaR-I on AECs was reduced whereas that of TbetaR-II was unaltered. However, 14 and 28 days after bleomycin injury, when there is decreased proliferation and induction of differentiation of type II AECs, there was a return of TbetaR-I expression on AECs. In contrast, TbetaR-I and TbetaR-II were observed on interstitial fibroblasts at all time intervals after bleomycin administration. Because both TbetaR-I and TbetaR-II are required for signal transduction, the reduction of TbetaR-I levels on the alveolar epithelium may alter the sensitivity of AECs to the antiproliferative effects of TGF-beta1 present in increased quantities following bleomycin injury. The loss of an antiproliferative response to TGF-beta1 may be important for the regeneration of the alveolar epithelium by proliferation while the expression of both receptors onfibroblasts would result in TGF-1 signaling for the synthesis of connective tissue proteins. Ourfindings suggest that during bleomycin-induced pulmonary fibrosis, the effects of TGF-beta1 on cells may be regulated by the expression of TbetaRs

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterised by subpleural fibrosis that progresses to involve all areas of the lung. The expression of transforming growth factor-beta1 (TGF-beta 1), a potent regulator of connective tissue synthesis, is increased in lung sections of patients with IPF. TGF-beta 1 is generally released in a biologically latent form (L-TGF-beta 1). Before being biologically active, TGF-beta must be converted to its active form and interact with both TGF-beta receptors type I and II (T beta R-I and T beta R-II). TGF-beta latency binding protein 1 (LTBP-1), which facilitates the release and activation of L-TGF-beta 1, is also important in the biology of TGF-beta 1. METHODS: Open lung biopsy samples from patients with IPF and normal controls were examined to localise T beta R-I, T beta R-II, and LTBP-1. Alveolar macrophages (AM) and bronchoalveolar lavage (BAL) fluid were examined using the CCL-64 bioassay to determine if TGF-beta is present in its active form in the lungs of patients with IPF. RESULTS: Immunoreactive L-TGF-beta 1 was present in all lung cells of patients with IPF except for fibroblasts in the subepithelial regions of honeycomb cysts. LTBP-1 was detected primarily in AM and epithelial cells lining honeycomb cysts in areas of advanced IPF. In normal lungs LTBP-1 immunoreactivity was observed in a few AM. AM from the upper and lower lobes of patients with IPF secreted 1.6 (0.6) fmol and 4.1 (1.9) fmol active TGF-beta, respectively, while AM from the lower lobes of control patients secreted no active TGF-beta (p< or =0.01 for TGF-beta in the conditioned media from AM obtained from the lower lobes of IPF patients v normal controls). The difference in percentage active TGF-beta secreted by AM from the lower lobes of patients with IPF and the lower lobes of control patients was significant (p< or =0.01), but the difference between the total TGF-beta secreted from these lobes was not significant. The difference in active TGF-beta in conditioned media of AM from the upper and lower lobes of patients with IPF was also not statistically significant. BAL fluid from the upper and lower lobes of patients with IPF contained 0.7 (0.2) fmol and 2.9 (1.2) fmol active TGF-beta, respectively (p< or =0.03). The percentage of active TGF-beta in the upper and lower lobes was 17.6 (1.0)% and 78.4 (1.6)%, respectively (p< or =0.03). In contrast, BAL fluid from control patients contained small amounts of L-TGF-beta. Using immunostaining, both T beta R-I and T beta R-II were present on all cells of normal lungs but T beta R-I was markedly reduced in most cells in areas of honeycomb cysts except for interstitial myofibroblasts in lungs of patients with IPF. TGF-beta 1 inhibits epithelial cell proliferation and a lack of T beta R-I expression by epithelial cells lining honeycomb cysts would facilitate repair of the alveoli by epithelial cell proliferation. However, the presence of both T beta Rs on fibroblasts is likely to result in a response to TGF-beta 1 for synthesis of connective tissue proteins. Our findings show that biologically active TGF-beta 1 is only present in the lungs of patients with IPF. In addition, the effects of TGF-beta 1 on cells may be further regulated by the expression of T beta Rs. CONCLUSION: Activation of L-TGF-beta 1 and the differential expression of T beta Rs may be important in the pathogenesis of remodelling and fibrosis in IPF

Circular parietal defects from 3 to 12 mm in diameter were made in 45 6-month old skeletally mature guinea pigs, and animals were sacrificed after survival periods of 3 days to 12 weeks. The original defect was harvested in continuity with a rim of surrounding bone and the adjacent dura and pericranium. After 12 weeks, all 3 and 5 mm defects were completely covered by a bridge of bone, while residual defects were noted within the 8 and 12 mm wounds. Percentage of new bone formation was significantly higher within 3 mm defects, than in all larger defects at each time interval from 1 week on (P < .05), reaching a mean of 93% in 3 mm defects and remaining below a mean of 31% in the remaining defect sizes. Immunolocalization demonstrated an osteogenic front in which the osteoblasts stained strongly for all isoforms of TGF-beta, with the intensity decreasing after the majority of the defects had reossified; this front was located at the advancing bone edge of the defect as well as the endocranial side adjacent to the dura. In conclusion, isoforms of TGF-beta are upregulated during a limited 'window' of time corresponding to the period of calvarial reossification, and are localized to osteoblasts within an osteogenic front at the periphery and dural surfaces of the defects

Transforming growth factor-betas (TGF-beta) have been demontstrated to be upregulated during osteoblast function in vitro and during cranial suture fusion in vivo. The authors hypothesized that spontaneous reossification of calvarial defects was also associated with upregulation of TGF-beta. The present study was designed to (1) evaluate the concept of a critical-size defect within the calvaria in an adult guinea pig model and (2) investigate the association between the ossification of calvarial defects and TGF-beta upregulation. Paired circular parietal defects with diameters of 3 and 5 mm and single parietal defects with diameters of 8 or 12 mm were made in 45 six-month-old skeletally mature guinea pigs. Three animals per defect size were killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method using an image analysis system in which the thickness of new bone was taken into account. Immunohistochemistry was performed using rabbit polyclonal antibodies to localize TGF-beta1, -beta2, and -beta3. All specimens were photographed, and the intensity of immunostaining was graded based on subjective photographic assessment by three independent reviewers. No defect demonstrated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of new bone, indicating these defects to be less than critical size. However, new bone formation in the 5-mm defects never exceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reaching a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks, and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocalization demonstrated osteogenic fronts at the advancing bone edge and the endocranial side, in which the osteoblasts stained strongly for all isoforms of TGF-beta. The intensity of osteoblast expression waned considerably after the majority of the defect had reossified. These data indicate that histometric analysis based on cross-sectional area more accurately reflects the osteogenic potential of a cranial defect than does linear inspection of defect closure. Although the interpretation of immunolocalization studies is highly subjective, independent assessment by three reviewers indicates that isoforms of TGF-beta were upregulated during a limited 'window' of time corresponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts

The three mammalian isoforms of transforming growth factor-beta (TGF-beta1, -beta2, and -beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin carcinogenesis, normal human keratinocytes, early (31) and late (310) passage immortalized keratinocytes (HaCaT cells), and five HaCaT-ras clones exhibiting benign (A-5, I-7), malignant (II-4, A-5 RT1), and highly aggressive (A-5 RT3) tumourigenic phenotypes were examined for the expression of TGF-beta isoforms, by immunohistochemistry. This was performed under in vivo conditions, in surface transplants and subcutaneously growing tumours in nude mice. Generally, all tissues that formed keratinized epithelia demonstrated an immunostaining pattern similar to normal human skin. TGF-beta1 was localized to the upper differentiated layers, the stratum granulosum and corneum, in a perimembranous pattern, whereas TGF-beta2 and, weaker, TGF-beta3 immunostaining was present in all suprabasal layers of normal keratinizing epithelia. In contrast, non-keratinizing transplants of non-tumourigenic or highly aggressive cells showed little to no immunoreactivity for TGF-beta1. Whereas TGF-beta2 expression was moderate in the upper layers of non-tumourigenic epithelia, large tumour cells of the malignant HaCaT-ras clones, particularly at the invasion front, were strongly positive for TGF-beta2. TGF-beta3 immunostaining was most pronounced in the stroma of malignant tumours, implying its paracrine induction by the malignant tumour transplants. These results suggest differential functions for each TGF-beta isoform in epidermal carcinogenesis, such that TGF-beta1 is associated with the more differentiated state, TGF-beta2 with highly malignant and invading cells, and TGF-beta3 with tumour stroma formation and angiogenesis. Furthermore, the expression of TGF-betas by both early- and late-stage tumours implies that the isoforms may have distinct functions at different stages of malignancy, supporting their dual role in skin carcinogenesis.

OBJECTIVE: To evaluate the parameters that mediate fibrogenesis in chronic pancreatitis (CP). BACKGROUND: Connective tissue growth factor (CTGF), which is regulated by transforming growth factor beta (TGF-beta), has recently been implicated in skin fibrosis and atherosclerosis. In the present study, the authors analyzed the concomitant presence of TGF-beta1 and its signaling receptors-TGF-beta receptor I, subtype ALK5 (TbetaR-I(ALK5)), and TGF-beta receptor II (TbetaR-II)-as well as CTGF and collagen type I in the pancreatic tissue of patients undergoing surgery for chronic pancreatitis. PATIENTS AND METHODS: CP tissue samples were obtained from 40 patients (8 women, 32 men) undergoing pancreatic resection. Tissue samples of 25 previously healthy organ donors (12 women, 13 men) served as controls. The expression of TGF-beta1, TbetaR-I(ALK5), TbetaR-II, CTGF, and collagen type I was studied by Northern blot analysis. By in situ hybridization and immunohistochemistry, the respective mRNA moieties and proteins were localized in the tissue samples. RESULTS: Northern blot analysis showed that CP tissue samples exhibited concomitant enhanced mRNA expression of TGF-beta1 (38-fold), TbetaR-II (5-fold), CTGF (25-fold), and collagen type I (24-fold) compared with normal controls. In addition, TbetaR-I(ALK5) mRNA was increased in 50% of CP tissue samples (1.8-fold). By in situ hybridization, TGF-beta1, TbetaR-I(ALK5), and TbetaR-II mRNA were often seen to be colocalized, especially in the ductal cells and in metaplastic areas where atrophic acinar cells appeared to dedifferentiate into ductal structures. In contrast, CTGF was located in degenerating acinar cells and principally in fibroblasts surrounding these areas. Moreover, CTGF mRNA expression levels correlated positively with the degree of fibrosis in CP tissues. CONCLUSION: The concomitant overexpression of CTGF, collagen type I, TGF-beta1, and its signaling receptors in CP suggests that these proteins contribute to enhanced extracellular matrix synthesis and accumulation, resulting finally in the fibrogenesis observed in CP