Biomedical subjects

R C Hovey

Publications and source records attributed to R C Hovey.

13 recordsLinked to original sources

PubMed2003

Developmental and hormonal signals dramatically alter the localization and abundance of insulin receptor substrate proteins in the mammary gland.

Insulin receptor substrates (IRS) are central integrators of hormone, cytokine, and growth factor signaling. IRS proteins can be phosphorylated by a number of signaling pathways critical to normal mammary gland development. Studies in transgenic mice that overexpress IGF-I in the mammary gland suggested that IRS expression is important in the regulation of normal postlactational mammary involution. The goal of these studies was to examine IRS expression in the mouse mammary gland and determine the importance of IRS-1 to mammary development in the virgin mouse. IRS-1 and -2 show distinct patterns of protein expression in the virgin mouse mammary gland, and protein abundance is dramatically increased during pregnancy and lactation, but rapidly lost during involution. Consistent with hormone regulation, IRS-1 protein levels are reduced by ovariectomy, induced by combined treatment with estrogen and progesterone, and vary considerably throughout the estrous cycle. These changes occur without similar changes in mRNA levels, suggesting posttranscriptional control. Mammary glands from IRS-1 null mice have smaller fat pads than wild-type controls, but this reduction is proportional to the overall reduction in body size. Development of the mammary duct (terminal endbuds and branch points) is not altered by the loss of IRS-1, and pregnancy-induced proliferation is not changed. These data indicate that IRS undergo complex developmental and hormonal regulation in the mammary gland, and that IRS-1 is more likely to regulate mammary function in lactating mice than in virgin or pregnant mice.

Adipose Tissue↗

PubMed2003

Alternative splicing to exon 11 of human prolactin receptor gene results in multiple isoforms including a secreted prolactin-binding protein.

Endocrine and autocrine prolactin (PRL) exerts effects on normal breast and breast cancer cells, and high serum PRL is a poor prognostic factor for colorectal cancer. Here we tested the hypothesis that short isoforms of the PRL receptor (PRLR) in human tissue regulate the actions of PRL in cancer. Using 3' RACE we isolated five splice variants of the human PRLR (hPRLR), three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9 respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant-negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant-negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.

Alternative Splicing↗

PubMed2001

Paracrine action of keratinocyte growth factor (KGF) during ruminant mammogenesis.

Keratinocyte growth factor (KGF) is a stroma-derived mitogen mediating epithelial-stromal interactions. We investigated the role of KGF during epithelial-stromal interactions accompanying ruminant mammogenesis. Target-specificity of KGF was demonstrated in that KGF-stimulated proliferation of bovine mammary epithelial, but not ovine mammary stromal cells. Consistent with a paracrine function, 4.6, 2.4, 1.5 and 0.9 kb mRNA transcripts were expressed by bovine stromal, but not epithelial cells. Within the ovine mammary gland, 2.4 and 1.5 kb KGF mRNAs were expressed in the fat pad while only the 2.4 kb transcript was transcribed in parenchyma. The abundance of KGF mRNA was greater in the extra-parenchymal fat pad than in the contralateral epithelium-free fat pad prior to puberty, and was less in parenchyma than in the intact or epithelium-free fat pads. Ovariectomy tended to increase KGF transcription while estrogen reduced expression. Of several tissues, mammary parenchyma expressed a 2.4 kb mRNA while adipose tissues expressed a 1.5 kb transcript. These results demonstrate local and systemic regulation of KGF transcription and support a paracrine role for KGF during ruminant mammogenesis.

Animals↗

PubMed2001

Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland.

Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L-PRLR) and two of the three short forms (S1- and S3-PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2-PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L-PRLR mRNA. By contrast, S1-PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2-PRLR became undetectable during puberty. Stromal expression of S3-PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.

Adipose Tissue↗

PubMed2001

Feedback regulation of PRL secretion is mediated by the transcription factor, signal transducer, and activator of transcription 5b.

PRL secretion from the anterior pituitary gland is inhibited by dopamine produced in the tuberoinfundibular dopamine neurons of the hypothalamus. The activity of tuberoinfundibular dopamine neurons is stimulated by PRL; thus, PRL regulates its own secretion by a negative feedback mechanism. PRL receptors are expressed on tuberoinfundibular dopamine neurons, but the intracellular signaling pathway is not known. We have observed that mice with a disrupted signal transducer and activator of transcription 5b gene have grossly elevated serum PRL concentrations. Despite this hyperprolactinemia, mRNA levels and immunoreactivity of tyrosine hydroxylase, the key enzyme in dopamine synthesis, were significantly lower in the tuberoinfundibular dopamine neurons of these signal transducer and activator of transcription 5b-deficient mice. Concentrations of the dopamine metabolite dihydroxyphenylacetic acid in the median eminence were also significantly lower in signal transducer and activator of transcription 5b-deficient mice than in wild-type mice. No changes were observed in nonhypothalamic dopaminergic neuronal populations, indicating that the effects were selective to tuberoinfundibular dopamine neurons. These data indicate that in the absence of signal transducer and activator of transcription 5b, PRL signal transduction in tuberoinfundibular dopamine neurons is impaired, and they demonstrate that this transcription factor plays an obligatory and nonredundant role in mediating the negative feedback action of PRL on tuberoinfundibular dopamine neurons.

Animals↗

PubMed2001

Transcriptional regulation of vascular endothelial growth factor expression in epithelial and stromal cells during mouse mammary gland development.

Accompanying changes in the development and function of the mammary gland is the establishment of a vascular network of critical importance for lactogenesis and tumorigenesis. A potent angiogenic and permeability factor that regulates vascular development in association with epithelial-stromal interactions is vascular endothelial growth factor (VEGF). Analysis of VEGF transcription by RT-PCR revealed mRNA for all three VEGF isoforms (VEGF120, 164, 188) within the mammary gland of nulliparous females. During pregnancy the level of VEGF188 declined and became undetectable during lactation in association with the increased abundance of VEGF120 and VEGF164 mRNAS: All three isoforms were expressed at consistent levels within the cleared mammary fat pad throughout development. Furthermore, the presence of VEGF188 mRNA in omental adipose tissue at various stages established that VEGF188 is expressed specifically in adipose tissue within the mammary gland. Using 3T3-L1 preadipocytes it was demonstrated that VEGF188 mRNA transcription occurs as a late event during lipogenesis distinct from earlier induction of VEGF120 and VEGF164 mRNA during differentiation. In contrast, HC11 mammary epithelial cells only expressed mRNA for VEGF120 and VEGF164. Localization of VEGF mRNA and protein revealed that VEGF is expressed in stromal cells of the mammary gland in nulliparous females and then undergoes a transition to epithelial expression during lactation. By contrast, mRNA for the VEGF receptors, Flk-1 and Flt-1, localized to stromal cells within the mammary fat pad during virgin and gestational development and was expressed in the interstitial tissue basal to epithelial cells during lactation. Taken together, these results support the conclusion that VEGF is differentially transcribed by specific cell types within the mammary gland, and that under hormonal regulation it functions in an autocrine/paracrine manner.

Adipocytes↗

PubMed2000

C/EBPbeta (CCAAT/enhancer binding protein) controls cell fate determination during mammary gland development.

Deletion of the transcription factor CCAAT/enhancer binding protein (C/EBP)beta results in a severe inhibition of lobuloalveolar development in the mouse mammary gland. Because progesterone receptor (PR) is requisite for alveolar development, the expression of PR was investigated in C/EBPbeta-/- mice. Unexpectedly, the number of PR-positive cells, as well as the levels of PR mRNA, were elevated 3-fold in the mammary glands of C/EBPbeta-/- mice. Furthermore, in contrast to wild-type nulliparous mice, in which PR distribution shifted from a uniform to nonuniform pattern between 8-12 weeks of age, C/EBPbeta-/- mice exhibited uniform PR distribution throughout all stages of mammary development analyzed. No change in C/EBPbeta mRNA levels was observed in the mammary glands of PR-/- mice, suggesting that PR acts in a pathway either in parallel to or downstream of C/EBPbeta. The overexpression and disrupted cellular distribution of PR in C/EBPbeta-/- mice were coincident with a striking 10-fold decrease in cell proliferation after acute steroid hormone treatment, assayed by incorporation of bromodeoxyuridine. In wild-type mice, PR and bromodeoxyuridine-positive cells were adjacent to each other and rarely colocalized. No differences in the level or pattern of PR expression were observed in the uterus, suggesting that C/EBPbeta influences PR in a mammary-specific fashion. Together, these data suggest that C/EBPbeta may control cell fate decisions in the mammary gland through the appropriate temporal and spatial expression of molecular markers, such as PR, that induce the proliferation of alveolar progenitor cells via juxtacrine mechanisms.

Animals↗

PubMed2000

Progesterone induces side-branching of the ductal epithelium in the mammary glands of peripubertal mice.

Development of the functional secretory epithelium in the mammary gland of the female mouse requires the elongation of the anlage through the mammary fat pad to form the primary/secondary ductal network from which tertiary ductal side-branches and lobuloalveoli develop. In this study we examined the hormonal requirements for the spatial development of the primary/secondary epithelial network and tertiary side-branches by quantifying ductal growth and epithelial cell proliferation in normal and hormone-treated BALB/c mice between 21 and 39 days of age. In normal mice, an allometric increase in ductal length commenced at 31 days of age and resulted in completion of the primary/secondary ductal network by 39 days of age. Concurrent with this allometric growth was a significant increase in cellular proliferation in the terminal end-buds (TEBs) of the ductal epithelium from 29 days of age, as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. A level of cellular proliferation similar to that in the TEBs of 33-day-old control mice could be induced in the TEBs of 25-day-old mice following treatment for 1 day with estrogen (E), or progesterone (P) or both (E/P), indicating that both E and P were mitogenic for epithelial cells of the peripubertal TEBs. However, the period of allometric ductal growth in untreated mice did not correspond to an increase in serum E or P (which might have been expected during the estrous cycle). In addition, epithelial growth was not observed in mammary glands from 24-day-old mice that were cultured in vitro with E, P or E/P. In contrast to treatment with E, treatment with P promoted a dramatic increase, relative to control mice, in the number of tertiary branch points upon the primary/secondary ductal network. BrdU labeling of mammary glands from 24- 33-day-old mice pelleted with cholesterol (C), E, P or E/P confirmed the greater mitogenicity of P on the epithelial cells of the secondary/tertiary ducts as compared with C or E. Concurrent with these changes, localized progesterone receptor (PR) expression in clusters of cells in the ductal epithelium was associated with structures that histologically resembled early branch points from ductules. In conclusion, our results suggest that additional endocrine growth factor(s) other than E and P contribute to the development of the primary/secondary ductal network, and that P is responsible for the formation of tertiary side-branches in the mammary glands of mice during puberty.

Aging↗

PubMed2000

Preparation of an epithelium-free mammary fat pad and subsequent mammogenesis in ewes.

The objective of this study was to develop a surgical procedure for the preparation of an epithelium-free mammary fat pad (cleared mammary fat pad; CFP) in ewes. At 7 to 10 d of age, ewe lambs (n = 43, mean BW 9.2 +/- .2 kg at 14 d) were sedated and one mammary gland was locally anesthetized. An incision circumscribing the base of the teat was made and blunt dissection was performed through the extraneous mammary fat pad tissue to enable the parenchyma and teat to be wholly removed. Failure to completely remove the epithelium enabled it to regenerate and grow into the mammary fat pad. Mean diameter of the parenchymal rudiment at 7 to 10 d of age was 8.9 +/- .5 mm (range of 5 to 16 mm). The excision site was closed with wound clips and recovered lambs returned to their dams. The contralateral mammary gland remained intact, allowing it to undergo normal development. Live weight gain was unaffected by this procedure. Ewes were subsequently slaughtered in groups at various stages of prepuberty, puberty, gestation, and lactation. Of 39 operated glands recovered, only one demonstrated epithelial outgrowth within the CFP. Parenchyma within the contralateral, intact gland underwent phases of rapid growth in prepuberty, puberty, and late gestation and was capable of milk synthesis after steroid induction or parturition. Change in weight of the CFP paralleled that of the intact mammary gland to 100 d of pregnancy. Sham CFP surgery was performed on four additional ewes wherein the parenchyma was completely excised and immediately replaced. Sham-operated epithelium populated the mammary fat pad and synthesized milk that could be expressed from the teat. A CFP in sheep will be a useful model for future investigations into the local growth regulatory mechanisms associated with ruminant mammogenesis.

Animals↗

PubMed1999

Regulation of mammary gland growth and morphogenesis by the mammary fat pad: a species comparison.

The growth and morphogenesis of mammary parenchyma varies substantially between species and is regulated by an array of systemic and local factors. Central to this regulation is the mammary fat pad, a matrix of adipose and connective tissue capable of mediating hormone action and synthesizing an array of growth regulatory molecules. In this article we highlight differences between the morphological development of the mammary parenchyma in rodents, humans, and ruminant dairy animals, placing emphasis on differences in the cellular composition and structure of the mammary fat pad. While a great deal remains to be understood about the ability of stroma to locally regulate mammary development, the significance of its contribution is becoming increasingly apparent. The actions of several steroid and peptide hormones appear to be mediated by an array of growth factors, proteases and extracellular matrix components synthesized by constituents of the mammary fat pad. Further, mammary adipose tissue represents a significant store of lipid which, by itself and through its derivatives, could influence the growth of mammary epithelium in diverse ways. This review describes the integral role of the mammary fat pad during mammogenesis, emphasizing the point that species differences must be addressed if local growth and morphogenic mechanisms within the mammary gland are to be resolved.

Adipose Tissue↗

PubMed1998

Ontogeny and epithelial-stromal interactions regulate IGF expression in the ovine mammary gland.

Although the insulin-like growth factors (IGF-I and -II) have been implicated in the stimulation of mammogenesis, little is known of their regulation in the mammary gland. In this study we removed epithelial tissue from one of the two mammary glands of 1-week-old ewe lambs and examined IGF-I and -II mRNA expression during postnatal development in both the intact mammary gland and in the gland cleared of epithelial tissue. Expression of IGF-I mRNA was highest at 6 and 10 weeks of age, coincident with the prepubertal phase of rapid mammary growth, then declined and remained low until expression increased during late pregnancy. IGF-I mRNA was more abundant in the mammary fat pad adjacent to parenchyma (MFP) than in the contralateral fat pad that had been surgically cleared of epithelium (CFP). The level of IGF-II mRNA in parenchyma was highest at 1-23 weeks of age due to an increase in the abundance of specific mRNAs. Expression was lower in the fat pads, with generally higher levels in the intact MFP than the CFP, and in these tissues IGF-II expression was shown to increase with age between 6 and 23 weeks. We also investigated the influence of the ovary and estrogen on the expression of IGFs. While IGF-I mRNA abundance was unaffected by ovariectomy, exogenous estrogen resulted in higher levels of expression in the MFP of ovariectomized ewes and tended to increase its level in the parenchyma of intact ewes. Ovariectomy increased IGF-II mRNA within mammary parenchyma whereas estrogen suppressed levels in both the parenchyma and MFP. These findings demonstrate that IGF-I and -II mRNAs are expressed locally within the developing ovine mammary gland and are regulated by stage of ontogeny, ovarian hormones, and epithelial stromal interaction.

Adipose Tissue↗

PubMed1998

The proliferation of mouse mammary epithelial cells in response to specific mitogens is modulated by the mammary fat pad in vitro.

The ability of the murine mammary fat pad to directly stimulate the growth of mammary epithelial cells and to modulate the effects of various mammogenic agents has been investigated in a newly described, hormone- and serum-free coculture system. COMMA-1D mouse mammary epithelial cells were cultured for 5 or 7 d with various supplements in the absence or presence of epithelium-free mammary fat pad explants from virgin female BALB/c mice. Cocultured fat pad stimulated increases in the DNA content of COMMA-1D cultures by two- to threefold or six- to eightfold after 5 or 7 d, respectively. The mitogenic effect was additive to that of 10% fetal calf serum and could not be attributed to the release of prostaglandin E2 or synthesis of prostaglandins by epithelial cells. In addition, bovine serum albumin attenuated (P < 0.05) the mitogenic effect of cocultured mammary fat pad. Added alone, insulinlike growth factor-I, epidermal growth factor, and insulin increased (P < 0.05) total DNA of COMMA-1D cultures by 2.5-, 3.7-, and 2.3-fold, respectively. Cocultured mammary fat pad markedly interacted (P < 0.01) with these mitogens to yield final DNA values that were 21.2-, 13.3-, and 22.1-fold greater than in basal medium only. Associated with this proliferation was the formation of numerous domes above the COMMA-1D monolayer. There was no proliferative response to growth hormone or prolactin in the absence or presence of cocultured fat pad (P > 0.05). Whereas hydrocortisone did not alter cell number, it attenuated (P < 0.05) the mitogenic effect of cocultured mammary fat pad. These results indicate that the murine mammary fat pad is not only a direct source of mitogenic activity, but also modulates the response of mammary epithelial cells to certain mammogens.

Adipose Tissue↗

PubMed1998

The mammary fat pad.

The mammary fat pad is essential for development of the mammary epithelium, providing signals that mediate ductal morphogenesis and, probably, alveolar differentiation. The "cleared" fat pad is often used as a transplantation site. Considering the crucial role of the fat pad, its properties have received relatively little attention from researchers in the field. Some of the questions whose investigation is pertinent to understanding both normal mammary development and carcinogenesis are outlined in this commentary in the spirit of stimulating enquiry into this important subject. It is clear from a brief perusal of the available literature that until studies are specifically designed to clearly differentiate between functional effects of the fibrous and the adipose stroma, more substantive information about their differential effects on mammary development and tumorigenesis will not be forthcoming.

Adipose Tissue↗