William J. DeVito

Distribution of immunoreactive prolactin in the male and female rat brain: effects of hypophysectomy and intraventricular administration of colchicine

Immunohistochemical studies have identified immunoreactive prolactin (IR-PRL) in the hypothalamus and other areas of the rat brain. However, immunocytochemical techniques make it difficult to quantify the amount of antigen localized in a specific region. In this study, IR-PRL was extracted from selected regions of the rat brain, consisting of the median eminence, dorsal and ventral hypothalamus, thalamus, amygdalae, cerebellum, cortex, hippocampus, septum, pons-medulla, and olfactory lobes, and the concentrations of IR-PRL were determined by radioimmunoassay. Whereas IR-PRL was detected in all brain regions in both the male and the female rat brain, the concentrations of IR-PRL in the female rat were significantly greater than those measured in the corresponding region of the male rat brain. In the female rat, hypophysectomy significantly reduced, but did not eliminate, the concentration of IR-PRL in hypothalamus, amygdala, thalamus, and pons-medulla. In contrast, hypophysectomy did not affect the concentration of IR-PRL in any brain regions of the male rat. Injection of colchicine into the lateral ventricle decreased the concentration of IR-PRL in the median eminence and increased the concentration of IR-PRL in the ventral hypothalamus in male and female rats. In addition, extracted hypothalamic and pituitary IR-PRL displayed similar dilution curves in the PRL assay and elution patterns on Sephadex G-100. These data indicate that both the male and the female rat brain contains an IR-PRL-like material with physicochemical properties similar to those of pituitary PRL. This material is differentially distributed in the male and female brain and is found in greater concentrations in the female brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: altered expression of collagenase, cell growth-related, and mineralization-associated genes

Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone‐like matrix. During the initial growth period (days 1–10), addition of acidic FGF (100 μg/ml) to actively proliferating cells increased (P < 0.05) 3H‐thymidine uptake (2,515 ± 137, mean ± SEM vs. 5,884 ± 818 cpm/104 cells). During the second stage of maturation (days 10–15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16–29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14–29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF‐induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7–8) enhanced histone H4, osteopontin, type 1 collagen, and TGF‐β mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14–15) reactivated H4, osteopontin, type I collagen, and TGF‐β gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 μg/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose‐related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF‐β and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence.

Prolactin‐Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C‐Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL‐induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL‐induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1‐(‐5‐isoquinolinesulfonyl)‐2‐methylpiperazine, or polymyxin B blocked the PRL‐induced increase in cell number with IC50 values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α‐phorbol 12,13‐didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U‐shaped dose‐response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose‐response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL‐induced increase in cell number. Finally, TPA treatment decreased cell‐surface binding of 125I‐PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.

Stem cells and prenatal origin of breast cancer

The hypothesis that in utero exposure to pregnancy hormones, notably estrogens, is related to the occurrence of breast cancer in the offspring has been examined in a number of epidemiological and experimental studies. Many studies have provided direct or indirect evidence that supports the hypothesis of an intrauterine component in the origin of breast cancer. Human studies to examine the underlying biological mechanisms, however, have been limited. We review the likely role of stem cells in hormone-mediated carcinogenic process, particularly as intermediate steps between in utero exposure to hormones and breast cancer. We summarize also studies related to the assumptions of the hypothesis concerning in utero exposure. We propose the use of stem cell potential as a measurable variable of the ‘fertile soil’, a term that has been used to characterize the consequences of fetal exposure to intrauterine environment. We conclude by outlining a feasible population-based study that measures stem cell potential to explore mechanisms mediating the relation between in utero exposure to pregnancy hormones and breast cancer risk in the offspring.

Prolactin induced expression of glial fibrillary acidic protein and tumor necrosis factor-alpha at a wound site in the rat brain.

To determine if PRL stimulates astrocyte proliferation and cytokine expression in vivo, we examined the effect of PRL on the wound-induced increase in the expression of glial fibrillary acid protein (GFAP) and tumor necrosis factor-alpha (TNF-alpha) in the CNS. Low levels of GFAP detected by Western blot analysis were identified in the non-wounded controls. Five days after the infliction of the wound, the relative abundance of GFAP in the tissue surrounding the wound site was greater than those of intact controls. Injection of PRL into the wound site markedly increased GFAP expression in the hypothalamus. Western blot analysis failed to detect TNF-alpha in the hypothalamus of non-wounded animals. In contrast, TNF-alpha was easily detected in the hypothalamus of wounded rats, and was markedly increased in PRL injected animals. To confirm the PRL-induced increase in TNF-alpha levels, TNF-alpha levels in hypothalamic extracts were measured by bioassay. In non-wounded controls, low but detectable TNF-alpha levels were found in the hypothalamus by bioassay (0.13 +/- 0.02 ng/mg protein). Infliction of a hypothalamic wound markedly increased TNF-alpha levels to 1.4 +/- 0.3 ng/mg protein. Injection of PRL into the wound site resulted in a further increase in TNF-alpha levels to 11.4 +/- 2.6 ng/mg protein. Further, infliction of the hypothalamic wound increased hypothalamic PRL content and PRL mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prolactin receptor expression by estradiol in the female rat brain

PROLACTIN (PRL) receptors have been identified in many tissues, including the brain, but little is known about their distribution and regulation. In the female rat brain, ovariectomy significantly (p < 0.05) decreased PRL binding capacity, but not the affinity, in the hypothalamus and pons-medulla. Using reverse transcription and polymerase chain reaction (RT-PCR) amplification and Western blot analyses we found both the long and short forms of the PRL receptor mRNAs and proteins in the hypothalamus, pons-medulla and cortex in the female rat. Ovariectomy decreased the expression of short, but not the long form of the PRL receptor in the hypothalamus and pons-medulla, but not the cortex. Administration of estradiol (1.0 mg per 100 g b.w.) restored the PRL binding capacity, protein and mRNA levels of the short form of the receptor back to control levels. These results suggest that the expression and distribution of PRL receptors in the brain are differentially regulated in specific brain regions.

Stimulation of hypothalamic prolactin release by veratridine and angiotensin II in the female rat : effect of ovariectomy and estradiol administration

In the female rat immunoreactive prolactin (IR-PRL) has been identified in the hypothalamus and in other brain regions. Brain IR-PRL is not of pituitary origin and, based on polyacrylamide gel electrophoresis and peptide mapping, shares a high degree of sequence homology with its pituitary counterpart. We have previously shown that hypothalamic tissue can release IR-PRL in vitro when depolarized by potassium. In this study, we examined the release of IR-PRL from hypothalami obtained from intact and ovariectomized rats and incubated in the presence of veratridine (an alkaloid which depolarizes excitable membranes), angiotensin II, or thyrotropin-releasing hormone. Hypothalamic tissue spontaneously released IR-PRL, and this release was significantly increased by veratridine or angiotensin II in a dose-dependent manner. The specificity of the angiotensin-II-evoked IR-PRL release was demonstrated by the inhibitory effect of saralasin, an angiotensin II receptor antagonist, on hypothalamic IR-PRL release. Thyrotropin-releasing hormone (100 microM) had no effect on hypothalamic IR-PRL release. Ovariectomy decreased hypothalamic IR-PRL content and IR-PRL release in response to veratridine and angiotensin II. The effect of estradiol on hypothalamic IR-PRL content and release was also examined by obtaining hypothalami from ovariectomized rats injected with estradiol (1 microgram/day) or vehicle for 5 days. When compared with vehicle injected rats, administration of estradiol significantly increased the hypothalamic IR-PRL content (46 +/- 4 vs. 81 +/- 16 ng/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol inhibits prolactin-induced activation of the JAK/STAT pathway in cultured astrocytes.

Alcohol consumption has multiple effects in the central nervous system (CNS). Whereas, alcohol is an immunosuppressive drug the effect of alcohol on the neuroimmune system, remains unclear. In cultured astrocytes, prolactin (PRL) induces mitogenesis and the expression of inflammatory cytokines, including tumor necrosis factor‐α (TNFα). We have recently shown that whereas ethanol does not inhibit PRL receptor binding, it markedly inhibits PRL‐induced mitogenesis and TNFα secretion in cultured astrocytes. It is clear that PRL activates the tyrosine phosphorylation of several proteins, including members of a novel family of protein tyrosine kinases, the Janus Kinases (JAKs). The aims of this study were to characterize PRL‐induced activation of the JAK/STAT (signal transducers and activators of transcription) pathway, and to determine if ethanol affects JAK/STAT activation in cultured astrocytes. We found that PRL specifically increases the tyrosine phosphorylation of JAK2, but not JAK1, JAK3, or Tyk2, and the subsequent phosphorylation of STAT1α, STAT5a, and STAT5b. Preincubation of astrocytes with ethanol markedly inhibited phosphorylation of JAK2, STAT1α, STAT5a, and STAT5b. In PRL‐stimulated astrocytes, ethanol inhibited binding of nuclear proteins to oligonucleotides corresponding to the gamma‐interferon activated sequence (GAS). Further, ethanol blocked PRL‐induced increases in interferon regulatory factor‐1 (IRF‐1) mRNA, a PRL/cytokine inducible transcription factor involved in the regulation of a number of cytokine inducible genes. The inhibition of tyrosine phosphorylation by ethanol was not a general effect, however, as we found that ethanol increased basal and NGF‐induced tyrosine phosphorylation of extracellular signal‐activated protein kinase‐1 (ERK‐1). These data indicate that ethanol inhibits PRL‐induced tyrosine phosphorylation of the JAK/STAT pathway resulting in decreased nuclear GAS DNA binding and inhibition of the PRL inducible gene, IRF‐1. Thus, suggesting that ethanol‐induced inhibition of JAK2 phosphorylation may be one mechanism though which ethanol could alter the brains response to injury or infection. J. Cell. Biochem. 74:278–291, 1999.

Prolactin stimulation of protein kinase C activity in the rat hypothalamus

Stimulation of cultured hypothalamic slices with PRL causes a rapid translocation of a Ca2+/phospholipid dependent protein kinase from the cytosol to the membrane fraction. The translocation of PKC from the cytosol to the membrane occurred at physiological concentrations of PRL with a maximal response occurring at 10(-10) M. At concentrations above this, there was less PKC activity translocated from the cytosol to the membrane. When injected into the medial preoptic area of the hypothalamus, PRL resulted in a similar translocation of PKC activity. These data clearly indicate that PRL can activate PKC in the rat hypothalamus, and suggest that PKC may be one of the transmembrane signaling mechanisms involved in the regulation of brain function by prolactin.

Immunoreactive Prolactin in the Hypothalamus and Cerebrospinal Fluid of Male and Female Rats

Immunoreactive prolactin (ir-PRL) has been identified in the cerebrospinal fluid (CSF) and brain of the male and female rat. In this study we determined the concentration of ir-PRL in the CSF and hypothalamus under conditions known to increase or decrease serum PRL. Hypophysectomy (60 days) significantly decreased the concentration of ir-PRL in the CSF of male (4.9 +/- 0.7 vs. 3.0 +/- 0.3 ng/ml) and female (5.8 +/- 0.9 vs. 3.1 +/- 0.5 ng/ml) rats. However, the effect of long-term hypophysectomy on hypothalamic ir-PRL was gender-dependent. That is, in the male rat hypophysectomy did not affect the content of ir-PRL in the female rat, long-term hypophysectomy decreased the content of ir-PRL in the median eminence, ventral hypothalamus, and dorsal hypothalamus 37, 40, and 47%, respectively. Estradiol replacement to the hypophysectomized female rat normalized the content of ir-PRL in the median eminence (96 +/- 5.8 to 131 +/- 9.6 ng/mg protein), ventral hypothalamus (11 +/- 0.6 to 16.0 +/- 1.1 ng/mg protein), dorsal hypothalamus (4.7 +/- 0.4 to 8.6 +/- 0.4 ng/mg protein), and the concentration ir-PRL in the CSF (2.5 +/- 0.3 to 4.6 +/- 0.4 ng/ml). In intact female rats, administration of haloperidol induced a marked hyperprolactinemia, and significantly increased CSF ir-PRL (5.1 +/- 1.5 vs. 18.0 +/- 3.8 ng/ml). However, in the same rats, the content of ir-PRL in the median eminence was significantly decreased while the ir-PRL content in the ventral hypothalamus and dorsal hypothalamus was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)