Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

Pregnancy causes a decrease in the number and affinity of myometrial angiotensin II receptors.

The pressor response to exogenously infused angiotensin II is decreased in normal human and ovine pregnancy when compared to the nonpregnant state. This study was designed to determine the mechanism for decrease in target organ sensitivity by examining pregnancy-induced changes in the affinity and number of angiotensin II receptors in myometrium from pregnant and nonpregnant sheep. Tissues were obtained from four ovariectomized sheep given 50 micrograms estradiol-17 beta on days 3 and 5 postsurgery and from four pregnant sheep (mean gestational age 136 days). Binding of 125I-angiotensin II was determined in particulate fractions of myometrium prepared by differential centrifugation. The 125I-angiotensin II binding to myometrial preparations was specific, saturable, and linear with protein concentration. In the nonpregnant group, binding capacity for angiotensin II in myometrium was 502 +/- 76 fmol/mg protein, whereas in the pregnant group it was 184 +/- 65 fmol/mg protein (P less than .01). Receptor affinity also was less (Kd 1.72 +/- 0.39 nM) during pregnancy compared with the nonpregnant sheep uterus (3.9 +/- 0.80 nM; P less than .01). These data support the hypothesis that the decrease in target organ sensitivity to exogenously administered angiotensin II during pregnancy in human and ovine species may be secondary to a decrease in the total number and affinity of specific angiotensin II receptors.

Hormonal control of phospholipid methylation in uterine luminal epithelial cells during uterine sensitivity to deciduogenic stimuli.

Estrogen secretion during pregnancy or pseudopregnancy defines a period of uterine sensitivity to deciduogenic stimuli, perhaps by altering the capacity of the luminal epithelium to respond to these stimuli. In several types of cells, the transduction of signals acting upon cell membranes appears to involve activation of the transmethylation of membrane phospholipids. To examine the role of membrane phospholipid methylation in the development and loss of uterine sensitivity, the capacity of luminal epithelial cells to incorporate 3H-methyl groups from [methyl-3H]methionine into phospholipid was determined during pseudopregnancy and after progestin and estrogen treatment of ovariectomized rats to induce uterine sensitivity. During pseudopregnancy, phospholipid transmethylation increased between days 3 and 4 and then decreased. Phospholipid transmethylation also decreased after estradiol treatment of progestin-pretreated ovariectomized rats. Lowest levels of methylation were observed on day 6 of pseudopregnancy or after 36 h of estradiol treatment when the uterus was no longer sensitive to deciduogenic stimuli. One of the earliest uterine responses to deciduogenic stimuli is an increase in vascular permeability. When phospholipid methylation was inhibited in luminal epithelial cells by administration of 3-deazaadenosine, the increase in vascular permeability following a deciduogenic stimulus was inhibited. These data suggest that the primary responses of uterine luminal epithelial cells to deciduogenic stimuli may involve phospholipid methylation and that decreases in cellular capacity for phospholipid methylation result in decreases in uterine sensitivity to these stimuli.

Biochemical responses of the luminal epithelium and uterine sensitization.

In our discussion of responses of the uterine epithelium to the blastocyst and their relationship to sensitization to deciduogenic stimuli, we have attempted to identify pieces of information that could be arranged into possible mechanisms. These choices and the limits of space resulted in a great deal of information that was not mentioned: changes in the charge and composition of the glycocalyx of the luminal epithelium, the morphology of the apical surface of the epithelium, steroid hormone receptor levels, epithelial protein synthesis, and most of the information obtained from species other than rats and mice. Despite whatever distortion that may have resulted from our reductions and simplifications of the mechanisms of implantation, it should be clear that blastocyst implantation involves communication not only between blastocyst and uterus, but also between various uterine tissues. Our understanding of implantation will be improved if we can identify the responses of individual tissues and the mechanisms of this communication. It seems likely that development and loss of uterine sensitivity controlled by progesterone and estradiol will depend upon separate biochemical responses in specific uterine tissues.

Uterine deoxyribonucleic acid synthesis during preimplantation in precursors of stromal cell differentiation during decidualization.

During early pseudopregnancy, DNA synthesis and mitosis in the uterine endometrial stroma precede the development of uterine sensitivity to deciduogenic stimuli. Progesterone redirects the effects of estradiol on endometrial DNA synthesis from the luminal epithelium to the stroma. To determine the time and hormonal control of preimplantation endometrial DNA synthesis, uterine cells were labeled with [3H] thymidine at specific times during early pseudopregnancy or after progestin and estrogen treatment of ovariectomized rats. The fate of these labeled cells after their decidualization was examined by separation of prelabeled deciduomal cells by velocity sedimentation at unit gravity, which separates cells by size. Stromal cells that synthesized DNA during early pseudopregnancy or in response to hormone treatment later differentiated into deciduomal cells. Rates of DNA synthesis increased on days 4 and 5 of pseudopregnancy, with greater incorporation occurring on day 4 in cells that differentiated into polyploid deciduomal cells. In ovariectomized rats, medroxyprogresterone acetate treatment for 15 h increased DNA synthesis in stromal cells that differentiated into diploid and tetraploid deciduomal cells. DNA synthesis increased further at 30 h before returning to basal levels at 48 h. After progestin pretreatment, estradiol treatment increased stromal DNA synthesis again with greater incorporation in cells differentiating into polyploid deciduomal cells. These data indicate that during early pseudopregnancy, both progesterone and estradiol control the DNA synthesis of endometrial stromal cells as a means of reprogramming these cells for the later growth and differentiation of decidualization.

Progestin increases cathepsin D synthesis in uterine luminal epithelial cells.

Early in blastocyst implantation, cells of the uterine luminal epithelium deteriorate and die in response to the presence of the blastocyst. Destruction of the epithelial cells appears to depend on control of the autophagic activity and enzyme content of lysosomes in these cells. Concentrations of the lysosomal proteinase, cathepsin D, have been identified in luminal epithelial cells, and these studies examined changes in epithelial cathepsin D activity and their hormonal control during early pseudopregnancy in the rat. Cathepsin D activity in luminal epithelial cells increases during early pseudopregnancy to maximal levels at the time of sensitivity to deciduogenic stimuli. Rates of cathepsin D synthesis in luminal epithelial cells also increase during early pseudopregnancy, but neither enzyme activity nor rates of synthesis increase in stromal-myometrial tissues. In ovariectomized rats, progestins rather than estradiol increase cathepsin D activity and rates of synthesis in luminal epithelial cells. These studies suggest that cell death in the luminal epithelium during blastocyst implantation may depend in part on the accumulation of lysosomal cathepsin D in these cells in response to progesterone secretion during early pregnancy.

Estrogen receptor in deciduoma cells separated by velocity sedimentation.

Nuclear estrogen receptor levels were measured in fractions of dispersed deciduoma cells separated according to size by velocity sedimentation at unit gravity. Previous studies demonstrated that velocity sedimentation separated larger fully differentiated polyploid deciduoma cells from smaller diploid cells. Since levels of estrogen receptor measured in homogenates of deciduoma tissue decreased during the growth and differentiation of the deciduoma, lower levels of estrogen receptor per cell were anticipated in polyploid deciduoma cells. Although levels of estrogen receptor decreased slightly in all fractions of deciduoma cells from days 7-8 of pseudopregnancy, the larger polyploid cells had higher levels of estrogen receptor than smaller cells on both days. These results indicate that differentiation of endometrial stromal cells during decidualization involves a decrease in receptor levels in all deciduomal cell types rather than a specific decrease in the receptor levels of only fully differentiated deciduoma cells. (Endocrinology 108: 484, 1981)