Claudia Gentili

Docosahexaenoic acid prevents apoptosis of retina photoreceptors by activating the ERK/MAPK pathway.

Identifying the trophic factors for retina photoreceptors and the intracellular pathways activated to promote cell survival is crucial for treating retina neurodegenerative diseases. Docosahexaenoic acid (DHA), the major retinal polyunsaturated fatty acid, prevents photoreceptor apoptosis during early development in vitro, and upon oxidative stress. However, the signaling mechanisms activated by DHA are still unclear. We investigated whether the extracellular signal regulated kinase (ERK)/mitogen‐activated protein kinase (MAPK) or the phosphatidylinositol‐3‐kinase (PI3K) pathway participated in DHA protection. 1,4‐Diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophynyltio) butadiene (U0126), a specific MEK inhibitor, completely blocked the DHA anti‐apoptotic effect. DHA rapidly increased ERK phosphorylation in photoreceptors, whereas U0126 blocked this increase. U0126 hindered DHA prevention of mitochondrial depolarization, and blocked the DHA‐induced increase in opsin expression. On the contrary, PI3K inhibitors did not diminish the DHA protective effect. DHA promoted the early expression of Bcl‐2, decreased Bax expression and reduced caspase‐3 activation in photoreceptors. These results suggest that DHA exclusively activates the ERK/MAPK pathway to promote photoreceptor survival during early development in vitro and upon oxidative stress. This leads to the regulation of Bcl‐2 and Bax expression, thus preserving mitochondrial membrane potential and inhibiting caspase activation. Hence, DHA, a lipid trophic factor, promotes photoreceptor survival and differentiation by activating the same signaling pathways triggered by peptidic trophic factors.

Involvement of PI3‐kinase and its association with c‐Src in PTH‐stimulated rat enterocytes

Phosphoinositide‐3‐kinase (PI3K) is a lipid kinase, which phosphorylates the D3 position of phosphoinositides, and is known to be activated by a host of protein tyrosine kinases. PI3K plays an important role in mitogenesis in several cell systems. However, whether parathyroid hormone (PTH) affects the activity and functional roles of PI3K in intestinal cells remain to be determined. The objective of this study was to identify and characterize the PI3K pathway, and its relation to other non‐receptor tyrosine kinases in mediating PTH signal transduction in rat enterocytes. PTH dose‐ and time‐dependently increased PI3K activity with a peak occurring at 2 min. The tyrosine kinase inhibitor genistein, c‐Src inhibitor PP1 and two structurally different inhibitors of PI3K, LY294002 and wortmannin, suppressed PI3K activity dependent on PTH. Co‐immunoprecipitation analysis showed a constitutive association between c‐Src and PI3K, which was enhanced by PTH treatment, suggesting that the cytosolic tyrosine kinase forms an immunocomplex with PI3K probably via the N‐SH2 domain of the p85α regulatory subunit. In response to PTH, tyrosine phosphorylation of p85α was enhanced, effect that was abolished by PP1, the inhibitor of c‐Src kinase. PTH causes a rapid (0.5–5 min) phosphorylation of Akt/PKB, effect that was abrogated by PI3K inhibitors, indicating that in rat enterocytes, PI3K is an upstream mediator of Akt/PKB activation by PTH. We report here that PI3K is also required for PTH activation of the mitogen‐activated protein kinases ERK1 and ERK2. Taken together, the present study demonstrate, for the first time, that PTH rapidly and transiently stimulates PI3K activity and its down effector Akt/PKB in rat enterocytes playing c‐Src kinase a central role in PTH‐dependent PI3K activation and that PI3K signaling pathway contributes to PTH‐mediated MAPK activation. J. Cell. Biochem. 86: 773–783, 2002.

Characterization of PTH/PTHrP receptor in rat duodenum: Effects of ageing

In rat enterocytes, signaling through the paratyroid hormone (PTH)/PTH‐related peptide receptor type 1(PTHR1) includes stimulation of adenylyl cyclase, increases of intracellular calcium, activation of phospholipase C, and the MAP kinase pathway, mechanisms that suffer alterations with ageing. The purpose of this study was to evaluate whether an alteration at the level of the PTH receptor (PTHR1) is the basis for impaired PTH signaling in aged rat enterocytes. Western Blot analysis with a specific monoclonal anti‐PTHR1 antibody revealed that a 85 kDa PTH binding component, the size expected for the mature PTH/PTHrP receptor, localizes in the basolateral (BLM) and brush border (BBM) membranes of the enterocyte, being the protein expression about 7‐fold higher in the BLM. Two other bands of 105 kDa (corresponding to highly glycosylated, incompletely processed receptor form) and 65 kDa (proteolytic fragment) were also seen. BLM PTHR1 protein expression significantly decreases with ageing, while no substantial decrease was observed in the BBM from old rats. PTHR1 immunoreactivity was also present in the nucleus where PTHR1 protein levels were similar in enterocytes from young and aged rats. Immunohistochemical analysis of rat duodenal sections showed localization of PTHR1 in epithelial cells all along the villus with intense staining of BBM, BLM, and cytoplasm. The nuclei of these cells were reactive to the PTHR1 antiserum, but not all cells showed the same nuclear staining. The receptor was also detected in the mucosae lamina propria cells, but was absent in globets cells from epithelia. In aged rats, PTHR1 immunoreactivity was diffused in both membranes and cytoplasm and again, PTH receptor expression was lower than in young animals, while the cell nuclei showed a similar staining pattern than in young rats. Ligand binding to PTHR1 was performed in purified BLM. rPTH(1‐34) displaced [I125]PTH(1‐34) binding to PTHR1 in a concentration‐dependent fashion. In both, aged (24 months) and young (3 months) rats, binding of [I125]PTH was characterized by a single class of high‐affinity binding sites. The affinity of the receptor for PTH was not affected by age. The maximum number of specific PTHR1 binding sites was decreased by 30% in old animals. The results of this study suggest that age‐related declines in PTH regulation of signal transduction pathways in rat enterocytes may be due, in part, to the loss of hormone receptors. J. Cell. Biochem. 88: 1157–1167, 2003.

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3′,5′-cyclic AMP and Ca2+

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca(2+) in these cells, in the present study we evaluated the involvement of cAMP, Ca(2+) and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 microM), an AC activator, or Sp-cAMP (50-100 microM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca(2+) (EGTA 0.5 mM), chelation of intracellular Ca(2+) with BAPTA (5 microM), or blockade of L-type Ca(2+)-channels with verapamil (10 microM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca(2+) mobilizing agent thapsigargin, the Ca(2+) ionophore A23187, ionomycin and membrane depolarization with high K(+). Inclusion of the calmodulin inhibitor fluphenazine (50 microM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca(2+) play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca(2+) and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.

PTH stimulates PLCβ and PLCγ isoenzymes in rat enterocytes: influence of ageing

We previously reported that in rat duodenal cells (enterocytes), parathyroid hormone (PTH [1-34]: PTH) stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP(3)) and diacylglycerol (DAG) and that this mechanism is severely altered in old animals. In the present study, we show that PTH [1-34]-dependent IP(3) release in young rats was blocked to a great extent by an antibody against guanine nucleotide binding protein Galphaq/11, indicating that the hormone activates a beta isoform of PLC coupled to the alpha subunit of Gq/11. In addition, PTH rapidly (within 30 s, with maximal effects at 1 min) stimulated tyrosine phosphorylation of PLCgamma in a dose-dependent fashion (10(-10)-10(-7) M). The hormone response was specific as PTH [7-34] was without effects. The tyrosine kinase inhibitors, genistein (100 microM) and herbimycin (2 microM), suppressed PTH-dependent PLCgamma tyrosine phosphorylation. Stimulation of PLCgamma tyrosine phosphorylation by PTH [1-34] greatly decreased with ageing. PP1 (10 microM), a specific inhibitor of the Src family of tyrosine kinases, completely abolished PLCgamma phosphorylation. The hormone-induced Src tyrosine dephosphorylation, a major mechanism of Src activation, an effect that was blunted in old animals. These results indicate that in rat enterocytes PTH generates IP(3) mainly through G-protein-coupled PLCbeta and stimulates PLCgamma phosphorylation via the nonreceptor tyrosine kinase Src. Impairment of PTH activation of both PLC isoforms upon ageing may result in abnormal hormone regulation of cell Ca(2+) and proliferation in the duodenum.

Age-related decline in mitogen-activated protein kinase phosphorylation in PTH-stimulated rat enterocytes.

In the present study we analyzed whether parathyroid hormone (rPTH[1-34]; PTH) stimulates the tyrosine phosphorylation of the growth-related protein mitogen-activated protein (MAP) kinases (p42/44-MAPK), also known as extracellular signal-regulated kinases (ERK1/2), in duodenal enterocytes isolated from young (3months) and aged (24months) rats. Western blot analysis revealed that PTH rapidly stimulates MAPK phosphorylation. The hormone effects on MAPK were evident within 30s, peaking at 1min (4-fold). PTH response was dose-dependent (10(-11)-10(-7) M) with maximal stimulation achieved at 10(-9)-10(-8) M. PTH-induced MAPK phosphorylation was effectively suppressed by the tyrosine-kinase inhibitors, genistein (100microM) and herbimycin (2microM). Moreover, the tyrosine phosphorylation and activation of MAPK was dependent on Src kinase, since PP1 (10 and 20microM), a specific Src family tyrosine-kinase inhibitor, blocked PTH-induced MAPK activation. With aging, the response to PTH was significantly reduced. However, The amount of basal protein expression determined by Western blot analysis for MAPK was not different in the enterocytes from young and aged rats. In conclusion, the results obtained in this work expand our knowledge on the mechanism of action of PTH in duodenal cells, revealing that protein tyrosine phosphorylation is linked to the PTH regulation of enterocyte MAPK activation, and that this mechanism is impaired with aging. Understanding the molecular mechanisms for the age-related differences in PTH signaling will require more information about the subtle mechanisms that modulate the PTH receptor-MAPK signaling pathway.

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone‐related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco‐2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco‐2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP‐dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF‐1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of β‐catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c‐Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco‐2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco‐2 cells. J. Cell. Biochem. 115: 2133–2145, 2014.

Role of PTHrP in human intestinal Caco-2 cell response to oxidative stress

We have previously demonstrated that parathyroid hormone (PTH) induces apoptosis in human colon adenocarcinoma Caco-2 cells but the effects of its tumoral analog PTH-related peptide (PTHrP) in this cell line are still unknown. In the present work we investigated whether PTHrP, as PTH, is able to induce Caco-2 cell apoptosis or if it exerts protective effects under apoptotic conditions. Using Caco-2 cells cultured under serum deprivation in the presence or absence of PTHrP we demonstrated that, differently to PTH, its analog employed at the same concentration (10(-8)M) is not a pro-apoptotic hormone. Cells were exposed to an oxidative insult in the form of hydrogen peroxide to induce apoptosis, which leads to a 50% loss of cell viability determined by MTS assay, morphological changes observed under fluorescence microscopy and Western blot analysis. Herein we demonstrate, for the first time, that pre-treatment with PTHrP prior to H2O2 incubation, prevents cell death induced by the apoptotic inductor; and using specific inhibitors we evidenced that protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 mitogen-activated protein kinase (MAPK) mediate this anti-apoptotic effect. Also, we found that PTHrP decreases the pro-apoptotic protein BAX levels and increases the protein expression of the anti-apoptotic HSP27. Immunoblot analysis revealed that H2O2 increases the phosphorylation levels of AKT and MAPKs, exhibiting a cellular defense response; and consequently increases phospho-BAD levels. The H2O2-induced activation of protein kinases is reverted when cells are pre-treated with PTHrP. Altogether these results evidence a protective effect of PTHrP under apoptotic conditions in intestinal cells, which may be mediated by AKT and MAPKs.

PTH and phospholipase A2 in the aging process of intestinal cells

In this study we analyzed, for the first time, alterations in phospholipase A2 (PLA2) activity and response to parathyroid hormone (PTH) in rat enterocytes with aging. We found that PTH, rapidly stimulate arachidonic acid (AA) release in rat duodenal cells (+1‐ to 2‐fold), an effect that is greatly potentiated by aging (+4‐fold). We also found that hormone‐induced AA release in young animals is Ca2+‐dependent via cPLA2, while AA released by PTH in cells from aged rats is due to the activation of cPLA2 and the Ca2+‐independent PLA2 (iPLA2). In enterocytes from 3 months old rats, PTH induced, in a time and dose‐dependent fashion, the phosphorylation of cPLA2 on serine 505, with a maximun at 10 min (+7‐fold). Basal levels of cPLA2 serine‐phosphorylation were higher in old enterocytes, affecting the hormone response which was greatly diminished (+2‐fold at 10 min). cPLA2 phosphorylation impairment in old animals was not related to changes of cPLA2 protein expression and did not explain the substantial increase on PTH‐induced AA release with aging, further suggesting the involvement of a different PLA2 isoform. Intracellular Ca2+ chelation (BAPTA‐AM, 5 μM) suppressed the serine phosphorylation of cPLA2 in both, young and aged rats, demonstrating that intracellular Ca2+ is required for full activation of cPLA2 in enterocytes stimulated with PTH. Hormone effect on cPLA2 was suppressed to a great extent by the MAP kinases ERK 1 and ERK2 inhibitor, PD 98059 (20 μM), the cAMP antagonist, Rp‐cAMP, and the PKC inhibitor Ro31820 both, in young and aged animals. Enterocytes exposure to PTH also resulted in phospho‐cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Hormone‐induced enzyme translocation is also modified by aging where, in contrast to young animals, part of phospho‐cPLA2 remained cytosolic. Collectively, these data suggest that PTH activates in duodenal cells, a Ca2+‐dependent cytosolic PLA2 and attendant AA release and that this activation requires prior stimulation of intracellular ERK1/2, PKA, and PKC. cPLA2 is the major enzyme responsible for AA release in young enterocytes while cPLA2 and the Ca2+‐independent iPLA2, potentiate PTH‐induced AA release in aged cells. Impairment of PTH activation of PLA2 isoforms upon aging may result in abnormal hormone regulation of membrane fluidity and permeability and thereby affecting intestinal cell membrane function.