Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

Significance of Increased Tissue Transglutaminase in Hormone Refractory Prostate Cancer

The progression of hormone responsive to hormone refractory prostate cancer poses a major clinical challenge in the successful treatment of prostate cancer. The hormone refractory prostate cancer cells exhibit resistance not only to castrate levels of testosterone, but also to other therapeutic modalities and hence become lethal. Currently, there is no effective treatment available for managing this cancer. These observations underscore the urgency to investigate mechanism(s) that contribute to the progression of hormone-responsive to hormone-refractory prostate cancer and to target them for improved clinical outcomes. Tissue transglutaminase (TG2) is a multifunctional pro-inflammatory protein involved in diverse physiological processes such as inflammation, tissue repair, and wound healing. Its expression is also implicated in pathological conditions such as cancer and fibrosis. Interestingly, we found that the androgen-independent prostate cancer cell lines, which lacked androgen receptor (AR) expression, contained high basal levels of tissue transglutaminase. Inversely, the cell lines that expressed androgen receptor lacked transglutaminase expression. This attracted our attention to investigate the possible role this protein may play in the progression of prostate cancer, especially in view of recent observations that its expression is linked with increased invasion, metastasis, and drug resistance in multiple cancer cell types. The results we obtained were rather surprising and revealed that stable expression of tissue transglutaminase in androgen-sensitive LNCaP prostate cancer cells rendered these cells independent of androgen for growth and survival by silencing the AR expression. The AR silencing in TG2 expressing cells (TG2-infected LNCaP and PC-3 cells) was due to TG2-induced activation of the inflammatory nuclear transcription factor-kB (NF-kB). Thus, TG2 induced NF-kB was found to directly bind to the AR promoter. Importantly, TG2 protein was specifically recruited to the AR promoter in complex with the p65 subunit of NF-kB. Moreover, TG2 expressing LNCaP and PC-3 cells exhibited epithelial-to-mesenchymal transition, as evidenced by gain of mesenchymal (such as fibronectin, vimentin, etc.) and loss of epithelial markers (such as E-cadherin, b-catenin). Taken together, these results suggested a new function for TG2 and revealed a novel mechanism that is responsible for the progression of prostate cancer to the aggressive hormone-refractory phenotype.

MODELLING β2AR REGULATION

The β2 adrenergic receptor (β2AR) regulates smooth muscle relaxation in the vasculature and airways. Long- and Short-acting β-agonists (LABAs/SABAs) are widely used in treatment of chronic obstructive pulmonary disorder (COPD) and asthma. Despite their widespread clinical use we do not understand well the dominant β2AR regulatory pathways that are stimulated during therapy and bring about tachyphylaxis, which is the loss of drug effects. Thus, an understanding of how the β2AR responds to various β-agonists is crucial to their rational use. Towards that end we have developed deterministic models that explore the mechanism of drug- induced β2AR regulation. These mathematical models can be classified into three classes; (i) Six quantitative models of SABA-induced G protein coupled receptor kinase (GRK)-mediated β2AR regulation; (ii) Three phenomenological models of salmeterol (a LABA)-induced GRK-mediated β2AR regulation; and (iii) One semi-quantitative, unified model of SABA-induced GRK-, protein kinase A (PKA)-, and phosphodiesterase (PDE)-mediated regulation of β2AR signalling. The various models were constrained with all or some of the following experimental data; (i) GRK-mediated β2AR phosphorylation in response to various LABAs/SABAs; (ii) dephosphorylation of the GRK site on the β2AR; (iii) β2AR internalisation; (iv) β2AR recycling; (v) β2AR desensitisation; (vi) β2AR resensitisation; (vii) PKA-mediated β2AR phosphorylation in response to a SABA; and (viii) LABA/SABA induced cAMP profile ± PDE inhibitors. The models of GRK-mediated β2AR regulation show that plasma membrane dephosphorylation and recycling of the phosphorylated β2AR are required to reconcile with the measured dephosphorylation kinetics. We further used a consensus model to predict the consequences of rapid pulsatile agonist stimulation and found that although resensitisation was rapid, the β2AR system retained the memory of prior stimuli and desensitised much more rapidly and strongly in response to subsequent stimuli. This could explain tachyphylaxis of SABAs over repeated use in rescue therapy of asthma patients. The LABA models show that the long action of salmeterol can be explained due to decreased stability of the arrestin/β2AR/salmeterol complex. This could explain long action of β-agonists used in maintenance therapy of asthma patients. Our consensus model of PKA/PDE/GRK-mediated β2AR regulation is being used to identify the dominant β2AR desensitisation pathways under different therapeutic regimens in human airway cells. In summary our models represent a significant advance towards understanding agonist-specific β2AR regulation that will aid in a more rational use of the β2AR agonists in the treatment of asthma.

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

$\beta\sb2$-adrenergic receptor desensitization, internalization and phosphorylation in response to full and partial agonists

The objective of this study is to test the hypothesis that partial agonists produce less desensitization because they generate less of the active conformation of the $\beta\sb2$-adrenergic receptor ($\beta$AR) (R*) and in turn cause less $\beta$AR phosphorylation by beta adrenergic receptor kinase ($\beta$ARK) and less $\beta$AR internalization. In the present work, rates of desensitization, internalization, and phosphorylation caused by a series of $\beta$AR agonists were correlated with a quantitative measure, defined as coupling efficiency, of agonist-dependent $\beta$AR activation of adenylyl cyclase. These studies were preformed in HEK-293 cells overexpressing the $\beta$AR with hemagglutinin (HA) and 6-histidine (6HIS) epitopes introduced into the N- and C-termini respectively. Agonists chosen provided a 95-fold range of coupling efficiencies, and, relative to epinephrine, the best agonist, (100%) were fenoterol (42%), albuterol (4.9%), dobutamine (2.5%) and ephedrine (1.1%). At concentrations of these agonists yielding $>$90% receptor occupancy, the rate and extent of the rapid phase (0-30 min) of agonist induced desensitization of adenylyl cyclase followed the same order as coupling efficiency, that is, epinephrine $\ge$ fitnoterol $>$ albuterol $>$ dobutamine $>$ ephedrine. The rate of internalization, measured by a loss of surface receptors during desensitization, with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like desensitization and internalization, $\beta$AR phosphorylation exhibited a dependency on agonist strength. The two strongest agonists epinephrine and fenoterol provoked 11 to 13 fold increases in the level of $\beta$AR phosphorylation after just 1 min, whereas the weakest agonists dobutamine and ephedrine caused only 3 to 4 fold increases in phosphorylation. With longer treatment times, the level of $\beta$AR phosphorylation declined with the strong agonists, but progressively increased with the weaker partial agonists. The major conclusion drawn from this study is that the occupancy-dependent rate of receptor phosphorylation increases with agonist coupling efficiencies and that this is sufficient to explain the desensitization, internalization, and phosphorylation data obtained.^ The mechanism of activation and desensitization by the partial $\beta$AR agonist salmeterol was also examined in this study. This drug is extremely hydrophobic and its study presents possibly unique problems. To determine whether salmeterol induces desensitization of the $\beta$AR its action has been studied using our system. Employing the use of reversible antagonists it was found that salmeterol, which has an estimated coupling efficiency near that of albuterol caused $\beta$AR desensitization. This desensitization was much reduced relative to epinephrine. Consistent with its coupling efficiency, it was found to be similar to albuterol in its ability to induce internalization and phosphorylation of the $\beta$AR. (Abstract shortened by UMI.) ^

ALTERATIONS IN HYPOTHALAMIC CONTENT OF LUTEINIZING HORMONE RELEASING HORMONE AND PITUITARY RESPONSIVENESS ASSOCIATED WITH TESTICULAR REGRESSION INDUCED BY PINEAL RELATED TREATMENTS IN THE GOLDEN HAMSTER

The adult male golden hamster, when exposed to blinding (BL), short photoperiod (SP), or daily melatonin injections (MEL) demonstrates dramatic reproductive collapse. This collapse can be blocked by removal of the pineal gland prior to treatment. Reproductive collapse is characterized by a dramatic decrease in both testicular weight and serum gonadotropin titers. The present study was designed to examine the interactions of the hypothalamus and pituitary gland during testicular regression, and to specifically compare and contrast changes caused by the three commonly employed methods of inducing testicular regression (BL,SP,MEL). Hypothalamic LHRH content was altered by all three treatments. There was an initial increase in content of LHRH that occurred concomitantly with the decreased serum gonadotropin titers, followed by a precipitous decline in LHRH content which reflected the rapid increases in both serum LH and FSH which occur during spontaneous testicular recrudescence. In vitro pituitary responsiveness was altered by all three treatments: there was a decline in basal and maximally stimulatable release of both LH and FSH which paralleled the fall of serum gonadotropins. During recrudescence both basal and maximal release dramatically increased in a manner comparable to serum hormone levels. While all three treatments were equally effective in their ability to induce changes at all levels of the endocrine system, there were important temporal differences in the effects of the various treatments. Melatonin injections induced the most rapid changes in endocrine parameters, followed by exposure to short photoperiod. Blinding required the most time to induce the same changes. This study has demonstrated that pineal-mediated testicular regression is a process which involves dynamic changes in multiply-dependent endocrine relationships, and proper evaluation of these changes must be performed with specific temporal events in mind. ^

Regulation of sex hormone dependent growth by a serum borne inhibitor

This thesis project focused on understanding the basic process controlling cell proliferation in sex-steroid hormone dependent cancers. The availability of inculture models using cloned cell lines offers the greatest advantage for studying the control of this event. Incubation of cloned sex-hormone sensitive cells in medium containing increasing concentrations of sex-hormone stripped serum, results in a dose dependent growth inhibition; this inhibition is reversed by the addition of physiological concentrations of steroid hormones. The mechanisms explaining this phenomenon are not yet fully understood, but different theories propose the existence in serum of a sex hormone binding protein with growth inhibitory properties. We were able to identify a protein that specifically binds sex hormones in rat and horse serum with affinities 10-fold lower to the ones observed with the classic sex-hormone binding globulin (SHBG) in humans. Purification of this protein on a large scale Lowed a more detailed analysis. The putative sex-hormone binding protein has an apparent molecular weight of 386 KDa. SDS-PAGE with commassie staining of the purified product, displayed a pattern non-characteristic of SMG, but all bands cross-reacted with a commercial anti-SMG antibody in western analysis. Titrations of the purified product on cell proliferation assays using sex-hormone dependent lines, resulted in a dose dependent growth inhibition. This inhibition was reversed by the addition of sex hormones. Our results indicate that we have identified and purified a sex-hormone binding protein in serum with characteristics similar to SHBG and with cell growth inhibitory properties. ^

Nuclear functions of dynein light chain 1 in hormone action

It is widely accepted that the process of breast cancer tumorigenesis involves estrogen receptor-alpha (ER)-regulated stimulatory pathways, which feed into survival, cell cycle progression and proliferative response. Recent data from Kumar laboratory indicate that dynein light chain 1 (DLC1) plays a role in survival, motility and invasiveness, all of which are required for a successful tumorigenesis process. In the present research, we have discovered a mechanistic bidirectional regulatory link between the DLC1 and ER. We found that DLC1 facilitates ligand-induced ER transactivation involving the recruitment of the DLC1-ER complex to ER-target genes. To gain insights into the mechanism by which DLC1 regulates the ER pathway, we set out to identify novel DLC1-interacting proteins. Among other proteins, we identified KIBRA and Ciz1 as two novel DLC1-interacting proteins. We found that the KIBRA-DLC1 complex is recruited to ER-responsive promoters, and that KIBRA-DLC1 interaction is needed for the recruitment of ER to its targets as well as for ER's transactivation function. Finally, we found that KIBRA utilizes its histone H3interacting glutamic acid-rich region to regulate the transactivation activity of ER. During the course of this work, we also discovered that DLC1 interacts with Cdk2 and Ciz1, and such interactions play a direct accelerating role in the G1-S transition of breast cancer cells. While delineating the role of Ciz1 in hormone-responsive cancer cells, we found that Ciz1 is an estrogen-responsive gene, and acts as a co-regulator of ER. Accordingly, Ciz1 overexpression in breast cancer cells conferred estrogen hypersensitivity, promoted the growth-rate, anchorage-independency and tumorigenic properties. Collectively, findings made during the course of the present dissertation research introduced two new molecular players in the action of ER in breast cancer cells, with a particular focus on cell cycle progression and ER-chromatin target regulation. In addition, findings presented here provide novel mechanistic insight about the contribution of DLC1 and its interacting proteins in amplifying the hormone action and promoting the process of breast cancer tumorigenesis. ^

Hormone therapy and physical activity as predictors of lung cancer risk and survival: The California Teachers Study

Cigarette smoking is responsible for the majority of lung cancer cases worldwide; however, a proportion of never smokers still develop lung cancer over their lifetime, prompting investigation into additional factors that may modify lung cancer incidence, as well as mortality. Although hormone therapy (HT), physical activity (PA), and lung cancer have been previously examined, the associations remain unclear. This study investigated exposure to HT and PA that may modulate underlying mechanisms of lung cancer etiology and progression among women by using existing, de-identified data from the California Teachers Study (CTS).^ The CTS cohort, established in 1995–1996, has 133,479 active and retired female teachers and administrators, recruited through the California State Teachers Retirement System, and followed annually for cancer diagnosis, death, and change of address. Each woman enrolled in the CTS returned a questionnaire covering a wide variety of issues related to cancer risk and women's health, including recent and past HT use and physical activity, as well as active and environmental cigarette smoke exposure. Complete data to assess the associations between HT and lung cancer risk and survival were available for 60,592 postmenopausal women. Between 1995 and 2007, 727 of these women were diagnosed with invasive lung cancer; 441 of these died. Complete data to assess the associations between PA and lung cancer risk and survival were available for 118,513 women. Between 1995 and 2007, 853 of these women were diagnosed with invasive lung cancer; 516 of these died.^ After careful adjustment for smoking habits and other potential confounders, no measure of HT use was associated with lung cancer risk; however, any HT use (vs. no use) was associated with a decrease in lung-cancer-specific mortality. Specifically, among women who only used estrogen (E-only), decreases in lung cancer mortality were seen for recent use, but not for former use; no association was observed for estrogen plus progestin (E+P). Furthermore, among former users of HT, a statistically significant decrease in lung cancer mortality was observed for E-only use within 5 years prior to baseline, but not for E-only use >5 years prior to baseline. Neither long-term recreational PA nor recent recreational PA alone were associated with lung cancer risk; however, among women with a BMI<25 and ever smokers, high long-term moderate+strenuous PA was associated with a decrease in lung cancer risk. Women with non-local disease showed a decrease in lung cancer mortality associated with increasing duration of strenuous long-term activity, and 1.50-3.00 h/wk/y of recent moderate or recent strenuous PA. Long-term moderate PA was associated with decreased lung cancer mortality in never smokers, whereas recent moderate PA was associated with increased lung cancer mortality in current smokers. ^ Placing our findings in the context of the current literature, HT does not appear to be associated with lung cancer risk and previous studies reporting a protective effect of HT use on lung cancer risk may be subject to residual confounding by smoking. Looking at our findings regarding PA overall, the evidence still remains inconclusive regarding whether or not physical activity influence lung cancer risk or mortality. Our results suggest that recreational PA may associated with decreased lung cancer risk among women with BMI<25 and ever smoking-women; however, residual confounding by smoking should be strongly considered. To our knowledge, this is the first study to investigate lifetime recreational PA and lung cancer mortality among women. Our results contribute to the growing body of knowledge regarding non-smoking-related risk factors for lung cancer incidence and mortality among women. Given the potential clinical and interventional significance, further study and validation of these findings is warranted.^

Longitudinal and logistic analysis of endocrine hormone profiles, physical maturation, and candidate genes related to pubertal events in children

Children who experience early pubertal development have an increased risk of developing cancer (breast, ovarian, and testicular), osteoporosis, insulin resistance, and obesity as adults. Early pubertal development has been associated with depression, aggressiveness, and increased sexual prowess. Possible explanations for the decline in age of pubertal onset include genetics, exposure to environmental toxins, better nutrition, and a reduction in childhood infections. In this study we (1) evaluated the association between 415 single nucleotide polymorphisms (SNPs) from hormonal pathways and early puberty, defined as menarche prior to age 12 in females and Tanner Stage 2 development prior to age 11 in males, and (2) measured endocrine hormone trajectories (estradiol, testosterone, and DHEAS) in relation to age, race, and Tanner Stage in a cohort of children from Project HeartBeat! At the end of the 4-year study, 193 females had onset of menarche and 121 males had pubertal staging at age 11. African American females had a younger mean age at menarche than Non-Hispanic White females. African American females and males had a lower mean age at each pubertal stage (1-5) than Non-Hispanic White females and males. African American females had higher mean BMI measures at each pubertal stage than Non-Hispanic White females. Of the 415 SNPs evaluated in females, 22 SNPs were associated with early menarche, when adjusted for race ( p<0.05), but none remained significant after adjusting for multiple testing by False Discovery Rate (p<0.00017). In males, 17 SNPs were associated with early pubertal development when adjusted for race (p<0.05), but none remained significant when adjusted for multiple testing (p<0.00017). ^ There were 4955 hormone measurements taken during the 4-year study period from 632 African American and Non-Hispanic White males and females. On average, African American females started and ended the pubertal process at a younger age than Non-Hispanic White females. The mean age of Tanner Stage 2 breast development in African American and Non-Hispanic White females was 9.7 (S.D.=0.8) and 10.2 (S.D.=1.1) years, respectively. There was a significant difference by race in mean age for each pubertal stage, except Tanner Stage 1 for pubic hair development. Both Estradiol and DHEAS levels in females varied significantly with age, but not by race. Estradiol and DHEAS levels increased from Tanner Stage 1 to Tanner Stage 5.^ African American males had a lower mean age at each Tanner Stage of development than Non-Hispanic White males. The mean age of Tanner Stage 2 genital development in African American and Non-Hispanic White males was 10.5 (S.D.=1.1) and 10.8 (S.D.=1.1) years, respectively, but this difference was not significant (p=0.11). Testosterone levels varied significantly with age and race. Non-Hispanic White males had higher levels of testosterone than African American males from Tanner Stage 1-4. Testosterone levels increased for both races from Tanner Stage 1 to Tanner Stage 5. Testosterone levels had the steepest increase from ages 11-15 for both races. DHEAS levels in males varied significantly with age, but not by race. DHEAS levels had the steepest increase from ages 14-17. ^ In conclusion, African American males and females experience pubertal onset at a younger age than Non-Hispanic White males and females, but in this study, we could not find a specific gene that explained the observed variation in age of pubertal onset. Future studies with larger study populations may provide a better understanding of the contribution of genes in early pubertal onset.^