Joseph P. Grande

Role of Transforming Growth Factor-β in Tissue Injury and Repair

Abstract Transforming growth factor-β (TGFβ) belongs to a family of multifunctional polypeptides which regulate normal cell growth, development, and tissue remodeling following injury. The ability of cells to produce TGFβ or to respond to this growth factor via cell surface receptors is highly conserved throughout the animal kingdom. TGFβ is a potent growth inhibitor of many normal and transformed cell lines; abnormalities in TGFβ signaling have been linked to tumorigenicity. Disruption of the TGFβ1 gene in utero produces a wasting syndrome characterized by systemic inflammation, suggesting that this growth factor plays an important role in limiting the inflammatory response. TGFβ is a dominant mediator of the pathologic extracellular matrix accumulation that characterizes progression of tissue injury to end-stage organ failure. Recent studies directed towards characterization of the TGFβ genes, dissection of the mechanisms by which TGFβs are produced and activated, and identification of TGFβ signaling pathways have established the important roles that these family members play in cell and tissue homeostasis. In this overview, TGFβ structure-function relationships and their relevance to a few select models of tissue injury/wound repair are highlighted.Transforming growth factor-beta (TGFbeta) belongs to a family of multifunctional polypeptides which regulate normal cell growth, development, and tissue remodeling following injury. The ability of cells to produce TGFbeta or to respond to this growth factor via cell surface receptors is highly conserved throughout the animal kingdom. TGFbeta is a potent growth inhibitor of many normal and transformed cell lines; abnormalities in TGFbeta signaling have been linked to tumorigenicity. Disruption of the TGFbeta1 gene in utero produces a wasting syndrome characterized by systemic inflammation, suggesting that this growth factor plays an important role in limiting the inflammatory response. TGFbeta is a dominant mediator of the pathologic extracellular matrix accumulation that characterizes progression of tissue injury to end-stage organ failure. Recent studies directed towards characterization of the TGFbeta genes, dissection of the mechanisms by which TGFbetas are produced and activated, and identification of TGFbeta signaling pathways have established the important roles that these family members play in cell and tissue homeostasis. In this overview, TGFbeta structure-function relationships and their relevance to a few select models of tissue injury/wound repair are highlighted.

Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues

Vitamin D3, via its active metabolite 1,25-dihydroxyvitamin D3, plays a critical part in male and female reproduction in the rat. 1,25-Dihydroxyvitamin D3 activity is mediated by an intracellular receptor (VDR). VDR distribution in reproductive tissue has not been studied using antibodies against the receptor. We developed a polyclonal antibody against the VDR and used it to examine VDR distribution in male and female rat reproductive tissues. In rat testes, VDR epitopes were observed in seminiferous tubules, specifically in spermatogonia, Sertoli cells and spermatocytes. Spermatozoa stained faintly. Epithelial cells of the epididymis, seminal vesicles and prostate also expressed VDR epitopes. In the female rat reproductive tract, immunostaining for VDR was seen in ovarian follicles, specifically in granulosa cells. Weaker VDR immunostaining was observed in follicular thecal cells and in the ovarian stroma and germinal epithelium. Corpus luteal cells stained intensely for VDR. Epithelium of fallopian tubes and the uterus also contained VDR epitopes. Both nuclear and cytoplasmic VDR immunostaining was observed in male and female rat reproductive tissues. We conclude that the VDR is widely distributed in male and female reproductive tissues and that it is likely to mediate actions of 1,25-dihydroxyvitamin D3 in the tissues.

Effects of high-fat diet and/or body weight on mammary tumor leptin and apoptosis signaling pathways in MMTV-TGF-α mice

IntroductionObesity is a risk factor for postmenopausal breast cancer and is associated with shortened mammary tumor (MT) latency in MMTV-TGF-α mice with dietary-induced obesity. One link between obesity and breast cancer is the adipokine, leptin. Here, the focus is on diet-induced obesity and MT and mammary fat pad (MFP) leptin and apoptotic signaling proteins.MethodsMMTV-TGF-α mice were fed low-fat or high-fat diets from 10 to 85 weeks of age. High-Fat mice were divided into Obesity-Prone and Obesity-Resistant groups based on final body weights. Mice were followed to assess MT development and obtain serum, MFP, and MT.ResultsIncidence of palpable MTs was significantly different: Obesity-Prone > Obesity-Resistant > Low-Fat. Serum leptin was significantly higher in Obesity-Prone compared with Obesity-Resistant and Low-Fat mice. Low-Fat mice had higher MFP and MT ObRb (leptin receptor) protein and Jak2 (Janus kinase 2) protein and mRNA levels in comparison with High-Fat mice regardless of body weight. Leptin (mRNA) and pSTAT3 (phosphorylated signal transducer and activator of transcription 3) (mRNA and protein) also were higher in MTs from Low-Fat versus High-Fat mice. Expression of MT and MFP pro-apoptotic proteins was higher in Low-Fat versus High-Fat mice.ConclusionThese results confirm a connection between body weight and MT development and between body weight and serum leptin levels. However, diet impacts MT and MFP leptin and apoptosis signaling proteins independently of body weight.

Intermittent Calorie Restriction Delays Prostate Tumor Detection and Increases Survival Time in TRAMP Mice

Prostate cancer is the most frequently diagnosed cancer in men. Whereas chronic calorie restriction (CCR) delays prostate tumorigenesis in some rodent models, the impact of intermittent caloric restriction (ICR) has not been determined. Here, transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were used to compare how ICR and CCR affected prostate cancer development. TRAMP mice were assigned to ad libitum (AL), ICR (2 wk 50% AL consumption followed by 2 wk pair feeding to AL consumption), and CCR (25% AL consumption) groups at 7 wk of age and followed until disease burden necessitated euthanasia or mice reached terminal endpoints (48 or 50 wk of age). Body weights fluctuated in response to calorie intake (P < 0.0001). ICR mice were older at tumor detection than AL (P = 0.0066) and CCR (P = 0.0416) mice. There was no difference for age of tumor detection between AL and CCR mice (P = 0.3960). Similar results were found for survival. Serum leptin, adiponectin, insulin, and IGF-I were all significantly different among the groups. These results indicate that the way in which calories are restricted impacts both time to tumor detection and survival in TRAMP mice, with ICR providing greater protective effect compared to CCR.

Effects of Intermittent and Chronic Calorie Restriction on Mammalian Target of Rapamycin (mTOR) and IGF-I Signaling Pathways in Mammary Fat Pad Tissues and Mammary Tumors

Chronic calorie restriction (CCR) prevents mammary tumor (MT) development in rodents. We reported that intermittent calorie restriction (ICR) provides greater protection than CCR in MMTV-TGF-α mice. The mammalian target of rapamycin (mTOR) pathway is involved in MT development. Here the impact of ICR versus CCR on proteins associated with mTOR signaling in mammary tissues and MTs from MMTV-TGF-α mice was determined. Mice were enrolled at 10 wk of age into ad libitum-fed (AL), CCR, and ICR groups and followed until 37/38 or 73/74 wk of age. Time points 37 and 73 followed 3 wk of 50% restriction for ICR mice, while 38 and 74 followed 1 wk of refeeding of ICR mice. Calorie restriction reduced serum IGF-I levels except for older CCR mice. At 37/38 wk, calorie restriction decreased mTOR, p70S6K, HIF-1, EGFR, and Erk protein activation and increased p4EBP1 and VEGF in mammary fat pads. At 73/74 wk, both modes of calorie restriction lowered IGF-I protein expression levels and Akt activation in MTs and mammary fat pads, and CCR increased mTOR, p70S6K, p4EBP1, and HIF-1 expression. ICR had inconsistent effects on these proteins in older mice. These results indicate that mTOR signaling proteins are modulated by age and type of calorie restriction.

Cross-sectional analysis of intermittent versus chronic caloric restriction in the TRAMP mouse

Previously we found that intermittent calorie restriction (ICR) delayed the age of prostate tumor detection and death in TRAMP mice in comparison to chronic calorie restricted (CCR) and ad libitum fed (AL) TRAMP mice.

Immunolocalization of Calcitriol Receptor, Plasma Membrane Calcium Pump and Calbindin-D28k in the Cornea and Ciliary Body of the Rat Eye

Epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump (PMCA) and the 28-kD vitamin-D-dependent Ca-binding protein (calbindin-D28k) were detected in sections of the albino rat eye using light microscopy and immunohistochemistry of paraffin-embedded tissues. Calcitriol receptor and calbindin-D28k epitopes were detected in both the inner and outer layers of the ciliary body epithelium. PMCA was present in both epithelial cell layers of the ciliary body but was most prominent in the inner layer. Corneal epithelium and endothelium also contained epitopes for calcitriol receptor, PMCA and calbindin-D28k. These Ca-regulatory proteins may play a role in the cellular physiology of the albino rat eye by maintaining appropriate intracellular and aqueous humor Ca concentrations.

Structure of the rat collagen IV promoter

We have isolated a 1.6 kb clone from a rat genomic library which contains the bidirectional collagen IV promoter, flanked by exons coding for the alpha 1 (IV) and alpha 2 (IV) collagen chains. There are at least two transcription start sites within both the alpha 1 (IV) and alpha 2 (IV) collagen genes. Rat mesangial cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing segments of the promoter and 5 flanking region, in both the alpha 1 (IV) and alpha 2 (IV) orientations. Our results suggest that transcriptional efficiency of the bidirectional promoter is more efficient in the alpha 2 (IV) direction than in the alpha 1 (IV) direction.

Renal Cell Cancer

A variety of tumors may arise in the kidneys, which vary widely in clinical significance. In adults, the most common form of malignant renal tumor is the adenocarcinoma, which arises from tubular epithelium. In part because the kidneys are located in the retroperitoneum, renal cell adenocarcinomas are characterized by a lack of early clinical warning signs; a substantial proportion of patients with renal cancer present with disseminated metastases. At present, surgical removal of renal cell adenocarcinomas is the only effective means of curing the disease, as these tumors are resistant to standard chemotherapy and radiation therapy. A potential role for immunotherapy in treating advanced renal cell cancer is being evaluated. Increased utilization of radiologic imaging techniques has led to the identification of small renal lesions in asymptomatic patients. Early detection of renal tumors has prompted the development of objective criteria for the classification of renal tumors of low and high metastatic potential and the establishment of uniformly accepted pathologic features of prognostic significance. Studies of patients with inherited predisposition to developing renal cell cancer have greatly contributed to our understanding of the pathogenesis of renal cell carcinoma. Inherited mutations in different genes predispose to the development of histologically distinct types of renal cancer. These studies provide support for the inclusion of genetic alterations in the histopathologic classification of renal tumors..