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Stromally derived keratinocyte growth factor (KGF) can play an important role in mammary gland development as a mesenchymal/stromal mediator of epithelial growth and morphogenesis. However, the possible coordinate regulation of mammary gland development by mammogenic hormones and KGF is unexplored. In these studies, the direct effect of mammogenic hormones on KGF-mediated mammary epithelial mitogenesis and expression of the KGF receptor was examined using primary mouse mammary epithelium growing in serum-free, collagen gel cell culture. Addition of KGF produced an average 7-fold increase in cell number after 10 days of culture. This effect of KGF was further increased in the presence of PRL (9-fold) or progesterone (P; 15-fold), with the combination of P and PRL (22-fold) producing the strongest synergistic stimulation. Estrogen did not show any additional stimulation of growth either alone or in combination with PRL and/or P. Ribonuclease protection analysis showed that epithelial cells grown in medium supplemented with P, but not PRL or estrogen, exhibited a 10-fold higher steady state level of KGF receptor (KGFR) messenger RNA (mRNA). KGFR expression was not induced by short term P exposure, suggesting an effect on mRNA stability rather than transcriptional activation. Time-course studies showed that an early decrease in the level of KGFR mRNA in basal cultures was significantly reduced by P addition. Measurement of RNA turnover after actinomycin D treatment showed that P increased the t(1/2) of KGFR mRNA compared with basal medium. Thus, P and PRL may differentially potentiate the direct mitogenic effect of KGF: P partly by elevation of the level of KGFR mRNA, and PRL principally by intracellular pathways not affecting KGFR expression.
Embryologically, the urinary bladder is formed from endodermally derived epithelial cells and mesenchymal cells from the urogenital sinus and allantois. Experimentally, we have shown that bladder mesenchyme differentiates into bladder smooth muscle via an unknown signaling mechanism that originates from the urothelium. It is hypothesized that this signaling between the cellular types, occurs via growth factors. Evidence supporting this hypothesis is that a number of known growth factors, such as TGF beta 2 and 3, KGF and TGF alpha, as well as their receptors are regulated as a function of bladder development and are also modulated during experimental bladder outlet obstruction. Furthermore, growth factors most likely affect extracellular matrix degradative proteins which play a role in bladder remodeling during development, as well as in partial outlet obstruction. There is certainly impressive cellular communication that occurs during development and also occurs postnatally; such as during bladder injury. We have recently shown that KGF is directly responsible for the proliferation of urothelium during bladder injury. This normally quiescent cell, which in humans turns over once every six months to a year when injured, has the incredible ability to immediately proliferate covering the exposed areas of bladder muscle and submucosa. This proliferation is due to the direct effects of KGF, a classic paracrine growth factor which is secreted by the stromal compartment of the bladder and acts directly on the urothelium which harbors the receptor. The bladder also has an uncanny ability to regenerate. In a model to study the basic science behind bladder regeneration, a partial cystectomy was performed and an acellular tissue matrix devoid of all cellular elements was sutured to the defect. Within four days, the urothelium completely covered the acellular matrix, and within two weeks native smooth muscle was seen streaming into the acellular matrix in association with a new epithelium. It is hypothesized that cellular interactions between the epithelium and the mesenchyme, as we have shown in bladder differentiation, are encouraging the new growth of smooth muscle. For the bladder to be a safe and effective storage chamber the ideal cellular lining should be urothelium. Cells from the gastrointestinal are not optimal for this purpose since they either secrete or absorb electrolytes. We believe that the cellular interactions that occur between the urothelium and the foreign intestinal stroma will in time change the phenotype of the urothelium. Newer strategies for bladder replacement which take into account cellular signaling are critical for our young patients with neurogenic bladder disorders.
INTRODUCTION - Surgical and traumatic injuries to the bladder initiate a complex series of biological processes that result in wound healing. This process involves cellular proliferation, migration and differentiation; removal of damaged tissue; and production of extracellular matrix all of which may be controlled by growth factors. In skin, keratinocyte growth factor (KGF) is induced following incisional injury. We hypothesize that in bladder wound healing KGF and other growth factors are induced to modulate tissue repair.
METHODS - We have created a model of surgical bladder injury in the rodent. At 12, 24 and 48 hrs and 5 and 7 days after injury, the bladder was bisected and total RNA extracted from the anterior or wounded half and posterior or non-wounded half. Histological analysis of the bladder wound was performed with Mason's Trichrome and immunohistochemistry against smooth muscle alpha actin. RNase protection assays were performed to examine the expression of KGF, transforming growth factor (TGF)alpha and TGF beta 2 and 3 as well as the receptors for KGF and epidermal growth factor (EGF). Lastly, the effects of the exogenous administration of KGF on the bladder was tested on neonatal mice by daily injections of 5 micrograms KGF per gram body weight for 5 days.
RESULTS - At 12 hours after injury KGF mRNA expression in the anterior wounded bladder half and posterior non-wounded bladder half was 8 and 6 times higher respectively, compared to unoperated control bladders. A similar response was seen for TGF alpha, where the 12 hour mRNA expression was 4.5 times higher in the anterior wounded bladder half and 3.5 times higher in the posterior non-wounded bladder half compared to unoperated control bladders. The nadir mRNA expression for both KGF and TGF alpha occurred at 7 days after bladder injury and was the same as in unoperated control bladders. EGFR mRNA expression was approximately 2 times higher in both the anterior wounded and posterior non-wounded bladder halves compared to the nadir levels which occurred at 24 hours after injury. TGF beta 2 and beta 3 mRNA levels did not significantly change in either the anterior wounded or posterior non-wounded bladder halves. Exogenous KGF stimulation resulted in a marked urothelial proliferation when compared to age matched control animals.
CONCLUSION - During the early phases of bladder wound healing (12-24 hours post injury), mRNA for KGF and TGF alpha increased, whereas TGF beta 2 and beta 3 and the KGFR and EGFR remain unchanged. Additionally, exogenous KGF has a direct effect on urothelial proliferation. KGF and TGF alpha warrant further study as potential mediators of bladder wound healing.
During fetal and neonatal development and experimental obstruction, the bladder wall undergoes changes in both the amount and composition of the urothelium, extracellular matrix, and smooth muscle. We hypothesize that cell-cell signaling among the different layers of the bladder wall mediates these changes. Growth factors likely to be involved in this process are keratinocyte growth factor (KGF) and transforming growth factor (TGF)-alpha, -beta 2, and -beta 3. Whole rodent bladders were analyzed by RNase protection assays for KGF, KGF receptor, TGF alpha, epidermal growth factor receptor, and TGF beta 2 and -beta 3 transcripts at Fetal Day 14 (before smooth muscle differentiation) and Fetal Day 18 (after smooth muscle differentiation), at birth, and 60 days postnatal. Growth factor transcripts were also analyzed in partially obstructed rodent bladders and in sham-operated animals. TGF beta 2 and -beta 3 mRNA expression decreased as a function of gestational age, whereas TGF alpha mRNA increased. KGF mRNA was low before smooth muscle differentiation at 14 days' gestation, then increased. The mRNA of receptors for KGF and EGF remained essentially unchanged throughout bladder development. In bladders subjected to partial urethral outlet obstruction, there was a 2-fold increase in mRNA for TGF beta 2, a 5-fold increase in TGF beta 3, and a 10-fold increase TGF alpha mRNA. In contrast, there was no change in transcripts for either KGF or receptors for KGF and epidermal growth factor. Immunohistochemical localization of the protein for these growth factors showed selective localization to the epithelium and/or smooth muscle for TGF beta 2 and -beta 3, whereas TGF alpha and the epidermal growth factor receptor localized throughout the bladder wall. In conclusion, growth factor mRNA expression is modulated in bladder development and obstruction, which implies a possible mechanistic role of growth factors for the observed changes in the bladder wall and extracellular matrix.
Tympanic membrane (TM) perforations heal by reepithelialization and fibrous layer proliferation. The rat TM model may be used to study growth factors that promote epithelialization and fibroblast proliferation, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). The authors previously evaluated the effects of FGF on tympanic membrane perforations and showed an enhanced rate of wound healing with preservation of normal structure and function. The same model was used to test keratinocyte growth factor (KGF, also called FGF-7). This growth factor has been shown to stimulate the migration and proliferation of keratinocytes. This is the first study investigating KGF in the tympanic membrane perforation model. Our results show that in contrast to FGF and EGF, KGF does not enhance the rate of wound healing, but rather results in a more organized wound repair process.