Leslie I. Gold

Enhanced expression of transforming growth factor β isoforms in pancreatic cancer correlates with decreased survival

BACKGROUND Transforming growth factor beta s (TGF-beta s) constitute a family of bifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. Perturbations in TGF-beta expression and function may lead to loss of negative constraints on cell growth. In this study, we examined TGF-beta expression in human pancreatic cancer. METHODS The distribution of TGF-beta isoforms in 60 human pancreatic cancers was examined using immunohistochemical, Northern blot, and in situ hybridization techniques. RESULTS Immunohistochemical analysis showed the presence of TGF-beta 1 (47% of tumors), TGF-beta 2 (42% of tumors), and TGF-beta 3 (40% of tumors) in the cancer cells. The presence of TGF-beta 2 was associated with advanced tumor stage (P < 0.05). Furthermore, there was a significant correlation between the absence of TGF-beta s in the tumors and longer postoperative survival. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas showed 11- (P < 0.001), 7- (P < 0.05), and 9-fold (P < 0.001) increases in the messenger RNA (mRNA) levels encoding TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively. By in situ hybridization, these mRNA moieties colocalized with their respective proteins in the cancer cells. CONCLUSIONS These findings show that human pancreatic cancers show increased levels of TGF-beta isoforms and enhanced TGF-beta mRNA expression and suggest that the presence of TGF-beta s in pancreatic cancer cells may contribute to disease progression.

Fibronectin: A review of its structure and biological activity

In 1948, MORRISON and co-workers demonstrated the co-precipitation of a high molecular weight plasma protein with cryofibrinogen following incubation in the cold 1. This protein was designated cold insoluble globulin (CIG). In 1973-1974 several laboratories using various techniques, made the independent observation that fibroblasts growing in tissue culture possessed a high molecular weight protein on their cell surface 2-7. CIG and this cell surface associated protein were established by immunological criteria to be identical a and subsequently by biochemical analysis to be very similar but to possess subtle differences in structure and function (see below). Fibronectin (Fn) is a widely accepted designation for both plasma and cell associated forms of this protein 9 and will be used throughout this review to represent both states of the protein. Differences between the two forms will be indicated where appropriate. Several reviews 1° 13 have extensively covered Fn and some degree of overlap has been unavoidable. However, the rapidly expanding attention and research effort devoted to this protein has allowed us to focus on more recent information and insights.

Calreticulin: non-endoplasmic reticulum functions in physiology and disease

Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT‐driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT‐null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER‐resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.—Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.‐M., Michalak, M., Murphy‐Ullrich, J. E. Calreticulin: non‐endoplamic reticulum functions in physiology and disease. FASEB J. 24, 665–683 (2010). www.fasebj.org

Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma.

BACKGROUND Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been suggested to influence tumor growth. They mediate their functions via specific cell surface receptors (type I ALK5 and type II TGF-beta receptors). The aim of this study was to analyze the roles of the three TGF-betas and their signaling receptors in human hepatocellular carcinoma (HCC). METHODS HCC tissue samples were obtained from 18 patients undergoing partial liver resection. Normal liver tissues from 7 females and 3 males served as controls. The tissues for histological analysis were fixed in Bouins solution and paraffin embedded. For RNA analysis, freshly obtained tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until used. Northern blot analysis was used in normal liver and HCC to examine the expression of TGF-beta1, -beta2, -beta3 and their receptors: type I ALK5 (TbetaR-I ALK5), type II (TbetaR-II), and type III (TbetaR-III). Immunohistochemistry was performed to localize the corresponding proteins. RESULTS All three TGF-betas demonstrated a marked mRNA overexpression in HCC in comparison with normal controls, whereas the levels of all three TGF-beta receptors showed no significant changes. Intense TGF-beta1, TGF-beta2, and TGF-beta3 immunostaining was found in hepatocellular carcinoma cells and in the perineoplastic stroma with immunohistochemistry, whereas no or mild immunostaining was present in the normal liver. For TbetaR-I ALK5 and TbetaR-II, the immunostaining in both HCC and normal liver was mild to moderate, with a slightly higher intensity in the normal tissues. CONCLUSION The upregulation of TGF-betas in HCC suggests an important role for these isoforms in hepatic carcinogenesis and tumor progression. Moreover, the localization of the immunoreactivity in both malignant hepatocytes and stromal cells suggests that TGF-betas act via autocrine and paracrine pathways in this neoplasm.

Studies in Cranial Suture Biology: Part I. Increased Immunoreactivity for TGF-β Isoforms (β1, β2, and β3) During Rat Cranial Suture Fusion†

The mechanisms involved in normal cranial suture development and fusion as well as the pathophysiology of craniosynostosis, a premature fusion of the cranial sutures, are not well understood. Transforming growth factor‐β isoforms (TGF‐β1, β2, and β3) are abundant in bone and stimulate calvarial bone formation when injected locally in vivo. To gain insight into the role of these factors in normal growth and development of cranial sutures and the possible etiology of premature cranial suture fusion, we examined the temporal and spatial expression of TGF‐β isoforms during normal cranial suture development in the rat. In the Sprague‐Dawley rat, only the posterior frontal cranial suture undergoes fusion between 12 and 22 days of age, while all other cranial sutures remain patent. Therefore, immunohistochemical analysis of the fusing posterior frontal suture was compared with the patent sagittal suture at multiple time points from the fetus through adult. Whereas the intensity of immunostaining was the same in the posterior frontal and sagittal sutures in the fetal rat, there was increased immunoreactivity for TGF‐β isoforms in the actively fusing posterior frontal suture compared with the patent sagittal suture starting 2 days after birth and continuing until approximately 20 days. There were intensely immunoreactive osteoblasts present during fusion of the posterior frontal suture. In contrast, the patent sagittal suture was only slightly immunoreactive. A differential immunostaining pattern was observed among the TGF‐β isoforms; TGF‐β2 was the most immunoreactive isoform and was also most strongly associated with osteoblasts adjacent to the dura and the margin of the fusing suture. Since the increased expression of TGF‐β2 during suture fusion suggested a possible regulatory role, recombinant TGF‐β2 was added directly to the posterior frontal and sagittal sutures in vivo to determine if suture fusion could be initiated. Exogenously added TGF‐β2 stimulated fusion of the ectocranial surface of the posterior frontal suture. These data provide evidence for a regulatory role for these growth factors in cranial suture development and fusion. Additionally, the intense immunostaining for TGF‐β2 in the dura mater underlying the fusing suture supports a role for the dura mater in suture fusion. It is possible that premature or excessive expression of these factors may be involved in the etiopathogenesis of craniosynostosis and that modulation of the growth factor profile at the suture site may have potential therapeutic value.

Connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis

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.

Immunolocalization of transforming growth factor beta 1, beta 2, and beta 3 and insulin-like growth factor I in premature cranial suture fusion.

The etiology of craniosynostosis remains unknown. The beta group of transforming growth factors (TGF-beta) and insulin-like growth factors (IGF-I and IGF-II) are known to induce new bone formation and, when added exogenously, cause accelerated closure of calvarial defects. The possible roles of these bone growth factors in premature cranial suture fusion in humans have not been explored. We analyzed a total of 20 cranial suture biopsy samples (10 synostotic and 10 normal) from 10 infants with single-suture craniosynostosis undergoing cranial vault remodeling. Using isoform-specific antibodies for TGF-beta 1, -beta 2, and -beta 3 and IGF-I, we demonstrated immunoreactivity of these growth factors were present in human cranial sutures; the TGF-beta 2 isoform was the most intensely immunoreactive. Most importantly, the TGF-beta isoforms and IGF-I showed more intense immunoreactivity in the actively fusing craniosynostotic sutures compared with the control patent sutures. Specifically, the TGF-beta isoforms and IGF-I were intensely localized in the osteoblasts synthesizing new bone at the suture margin. It is noteworthy that although the patent sutures were less immunoreactive for TGF-beta isoforms than fused sutures, there was a distinct pattern of the TGF-beta 3 isoform that was immunolocalized to the margin of the normal patent sutures. This suggests a possible role for TGF-beta 3 in maintaining cranial suture patency. The increased immunoreactivity of both TGF-beta 2 and IGF-I in the actively fusing sutures compared with the patent control sutures indicates that these growth factors may play a role in the biology underlying premature suture closure. To our knowledge, this is the first study showing the presence of TGF-beta 1, -beta 2, and -beta 3 and IGF-I in prematurely fusing human cranial sutures. In the future, manipulating the local expression of these growth factors at the suture site may enable plastic surgeons to modulate premature suture fusion.

Calreticulin Enhances Porcine Wound Repair by Diverse Biological Effects

Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds.

Enhanced expression of TGF-βs and their receptors in human acute pancreatitis

OBJECTIVES To determine which mechanisms are involved in pancreatic remodeling, repair, and fibrosis after acute necrotizing pancreatitis (NP) in humans. SUMMARY BACKGROUND DATA Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been implicated in the regulation and formation of extracellular matrix and fibrosis. They exert their functions by binding to specific receptors. In this study, we analyze the expression of TGF-beta1, TGF-beta2, and TGF-beta3 and their receptors type I (Tbeta-RI [ALK5]), type II (Tbeta-RII), and type III (Tbeta-RIII) in NP. PATIENTS Pancreatic tissue samples were obtained from 6 female and 8 male patients with a median age of 65 years (range, 37 to 77 years) undergoing surgery for NP. The median Ranson score of the patients was 6 (range, 2 to 9). The operation was performed a median 5.5 days (range, 4 to 17 days) after the onset of acute pancreatitis. Pancreatic tissue obtained from 12 previously healthy organ donors (6 male, 6 female; median age of 43 years) served as controls. METHODS The expression of TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), Tbeta-RII, Tbeta-RIII, and collagen type I mRNA was analyzed by Northern blot analysis. In addition, immunohistochemical analysis using polyclonal antibodies was performed to detect TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), and Tbeta-RII. RESULTS Northern blot analysis showed an increase in TGF-betas and their receptors in NP tissue samples compared with samples from normal controls. The increase was 3.5-fold for TGF-beta1 (p < 0.05), 2.7-fold for TGF-beta2 (p < 0.05), 3.5-fold for TGF-beta3 (p < 0.05), 10-fold for Tbeta-RI (ALK5) (p < 0.05), 5.7-fold for Tbeta-RII (p < 0.05), and 1.4-fold for Tbeta-RIII (not significant). Collagen type I mRNA was also markedly increased in NP samples and correlated with the level of TGF-betas. Immunohistochemical analysis demonstrated intense TGF-beta1, TGF-beta2, TGF-beta3, Tbeta-RI (ALK5), and Tbeta-RII immunoreactivity in the remaining acinar and ductal cells in most NP samples; in the normal control pancreas, there was weak to moderate immunoreactivity for these factors only in some acinar cells and a few ductal cells. CONCLUSION The marked increase in expression of TGF-betas and their signaling receptors Tbeta-RI (ALK5) and Tbeta-RII suggests a role for TGF-betas in the repair process after the onset of NP in humans and raises the possibility that TGF-betas might be involved in tissue remodeling and the fibrotic reaction that occurs in the pancreas after necrosis.

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the pancreatic parenchyma.