**7. E-RAF in pregnant rat uterus: significance of the findings**

Premkumar and Thampan (1995) examined the level of E-RAF in the uteri of pregnant rats during a full term of pregnancy. It was noticed that from day 1 of pregnancy the E-RAF titer in the uterus registered a steady increase. It reached an all time peak towards midpregnancy following which E-RAF level began to decline. The rate of decline was found to be very fast; two days before parturition the uterine E-RAF titer became virtually undetectable. It is known that progesterone is essential for maintaining the functional integrity of the pregnant uterus. The possibility, therefore, exists that the E-RAF titer is a reflection of the progesterone requirement of the pregnant uterus. The decline in E-RAF titer

Evolving Trends in Estrogen Receptor Biology 71

(1) Blood plasma (2) Target cell(3)E-RAF-GRA(E-RAF gene regulatory agent)(4)receptor for E-RAF-GRA(5)signal transduction events that succeed the receptor –GRA-interaction(6)E-RAF gene

Fig. 3. **A model that explains the role of an external factor, transported by the blood, on uterine E-RAF synthesis.** The factor recognizes its receptor on the target cell plasma membrane and induces signal transduction events which eventually terminate in enhanced

The model indicates that the regulatory factor,E-RAF gene regulatory agent(E-RAF-GRA) transported by the blood, binds to its receptor on the plasma membrane of the uterine cell. This interaction initiates a cascade of signal transduction events, ultimately leading to the enhancement in E-RAF gene expression in the nucleus and synthesis in the cytoplasm. The decline in the circulating presence of the factor, as it may happen during the second half of pregnancy, will be reflected in the decline in uterine E-RAF gene expression. Preliminary report has already been made on the presence of E-RAF in the goat uterine mitochondria (Praseetha & Thampan,2009). Possibly one of the major functions of E-RAF is to transport cholesterol to the mitochondria where it will be converted enzymatically to pregnenolone

E-RAF could play a role in the progression of mammary cancer. E-RAF II is a very active transcription factor and this molecular form of E-RAF represents more than 75%of total E-RAF population representing both E-RAFI and E-RAF II. If one assumes that the benign to malignant transformation of the mammary cancer is associated with enhanced expression of E-RAFII, that should be reflected in immunofluorescent detection of E-RAF in frozen

expression(7)synthesis of E-RAF in the cytoplasm.

expression of E-RAF gene.

and progesterone.

**8. E-RAF and mammary cancer** 

during the second half is again indicative of the need for progesterone withdrawal prior to parturition. The hypothetical presentation given in figure 2 takes into account the E-RAF titer in rat uterus during pregnancy.

Fig. 2. **A generalized plan of E-RAF titer in rat uterus during pregnancy.** Uterine tissue was collected from a group of rats everyday during the 21-day pregnancy term. The tissue samples were homogenized in the coating buffer (10mM carbonate and 40mM bicarbonate buffer, pH 9.6), and the homogenates were centrifuged at 10,000Xg for 15 minutes. To the supernatant an equal volume of a suspension of DEAE cellulose in coating buffer was added. After 30 minutes of incubation in ice, the DEAE cellulose flow through fraction was collected. An aliquot of this fraction was used for E-RAF estimation through ELISA. The data takes into account the potential existence of an intracellular threshold level of E-RAF. It is being postulated that, beyond this threshold E-RAF enters the blood and gets transported to the specified site that, under conditions where E-RAF titer is low releases the hypothetical factor which, upon binding to its receptor on the uterine cell membrane, initiates the signal transduction events leading to enhanced E-RAF gene expression.

During the negative regulatory mechanism the enhanced level of E-RAF could inhibit the release of the stimulatory factor, thereby suppressing the signal transduction process leading to E-RAF gene activation.

If one assumes that there is an upper limit (threshold) in the uterine level of E-RAF, beyond which the E-RAF enters the blood, it is possible to reconstruct a molecular event. E-RAF is probably transported by the blood to an anatomical site (brain?), which is responsible for enhancing uterine E-RAF gene expression through distinct signal transduction mechanisms mediated by a specific macromolecular agent. Possibly, the presence of E-RAF in circulation could inhibit the release of this mediator, the eventual result being the decline in E-RAF synthesis, leading to the final disappearance of the proteins prior to parturition. Figure 3 illustrates the proposed mechanism of action of this hypothetical regulator of E-RAF gene expression in uterine cells.

during the second half is again indicative of the need for progesterone withdrawal prior to parturition. The hypothetical presentation given in figure 2 takes into account the E-RAF

Fig. 2. **A generalized plan of E-RAF titer in rat uterus during pregnancy.** Uterine tissue was collected from a group of rats everyday during the 21-day pregnancy term. The tissue samples were homogenized in the coating buffer (10mM carbonate and 40mM bicarbonate buffer, pH 9.6), and the homogenates were centrifuged at 10,000Xg for 15 minutes. To the supernatant an equal volume of a suspension of DEAE cellulose in coating buffer was added. After 30 minutes of incubation in ice, the DEAE cellulose flow through fraction was collected. An aliquot of this fraction was used for E-RAF estimation through ELISA. The data takes into account the potential existence of an intracellular threshold level of E-RAF. It is being postulated that, beyond this threshold E-RAF enters the blood and gets transported to the specified site that, under conditions where E-RAF titer is low releases the hypothetical factor which, upon binding to its receptor on the uterine cell membrane, initiates the signal

During the negative regulatory mechanism the enhanced level of E-RAF could inhibit the release of the stimulatory factor, thereby suppressing the signal transduction process

If one assumes that there is an upper limit (threshold) in the uterine level of E-RAF, beyond which the E-RAF enters the blood, it is possible to reconstruct a molecular event. E-RAF is probably transported by the blood to an anatomical site (brain?), which is responsible for enhancing uterine E-RAF gene expression through distinct signal transduction mechanisms mediated by a specific macromolecular agent. Possibly, the presence of E-RAF in circulation could inhibit the release of this mediator, the eventual result being the decline in E-RAF synthesis, leading to the final disappearance of the proteins prior to parturition. Figure 3 illustrates the proposed mechanism of action of this hypothetical regulator of E-RAF gene

transduction events leading to enhanced E-RAF gene expression.

leading to E-RAF gene activation.

expression in uterine cells.

titer in rat uterus during pregnancy.

(1) Blood plasma (2) Target cell(3)E-RAF-GRA(E-RAF gene regulatory agent)(4)receptor for E-RAF-GRA(5)signal transduction events that succeed the receptor –GRA-interaction(6)E-RAF gene expression(7)synthesis of E-RAF in the cytoplasm.

Fig. 3. **A model that explains the role of an external factor, transported by the blood, on uterine E-RAF synthesis.** The factor recognizes its receptor on the target cell plasma membrane and induces signal transduction events which eventually terminate in enhanced expression of E-RAF gene.

The model indicates that the regulatory factor,E-RAF gene regulatory agent(E-RAF-GRA) transported by the blood, binds to its receptor on the plasma membrane of the uterine cell. This interaction initiates a cascade of signal transduction events, ultimately leading to the enhancement in E-RAF gene expression in the nucleus and synthesis in the cytoplasm. The decline in the circulating presence of the factor, as it may happen during the second half of pregnancy, will be reflected in the decline in uterine E-RAF gene expression. Preliminary report has already been made on the presence of E-RAF in the goat uterine mitochondria (Praseetha & Thampan,2009). Possibly one of the major functions of E-RAF is to transport cholesterol to the mitochondria where it will be converted enzymatically to pregnenolone and progesterone.
