Involvement of stress activated protein kinases (JNK and p38) in 1,25 dihydroxyvitamin D3-induced breast cell death

It has been previously demonstrated that 1,25 dihydroxyvitamin D₃ (1,25-D₃) exerts inhibitory effects in breast cancer cells. The aim of this study was to determine whether mitogen-activated protein kinase (MAPK) pathways are associated with 1,25-D₃-induced cell death in breast cancer. We used three breast cell lines which have different sensitivities to 1,25-D₃ treatment. Non-malignant MCF-12A cells were more sensitive to 1,25-D₃ treatment than malignant MCF-7 cells (growth inhibition IC₅₀ 75 nM vs. 100 nM, p < 0.001) while malignant MDA-MB-231 cells were resistant. Moreover, 1,25-D₃-induced apoptosis was caspase-dependent in MCF-12A cells and caspase-independent in MCF-7 cells. Following MAPK activation analysis, we found a significant activation of JNK in MCF-12A cells and malignant MCF-7 cells in response to 1,25-D₃ treatment. Furthermore, 1,25-D₃ treatment stimulated p38 activity in MCF-12A cells and in MCF-7 cells. ERK1/2 activity was unaffected by 1,25-D₃ treatment in all breast cells. Importantly, no increased MAPK activity was observed in MDA-MB-231 breast cancer cells which displayed resistance to 1,25-D₃-induced apoptosis. Utilising specific pharmacological inhibitors of JNK and p38, it was demonstrated that MCF-12A and MCF-7 cells were protected from death induced by 1,25-D₃. These results implicate JNK and p38 signalling in 1,25-D₃-induced cancer breast cell death.     

Early effect of 25-hydroxycholecalciferol (25-OH-D3) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) on the ability of parathyroid hormone (PTH) to elevate cyclic AMP of intact bone cells.

25-OH-D₃ and 1,25-(OH)₂-D₃ had no effects by themselves on the cyclic AMP levels of isolated bone cells but enhanced the stimulation seen following an exposure with submaximal concentrations of PTH for as little as 2 minutes. Preincubation with the 25-OH-D₃ or 1,25-(OH)₂-D₃ resulted in a time dependent decrease in the enhancement of PTH response over a 1 hr period. It is, therefore, suggested that cyclic AMP may be involved in some aspects of the action of vitamin D₃ derivatives on bone cells.     

Studies on calciferol metabolism: Metabolism of 25-hydroxy-Vitamin D3 by the chicken embryo

It is known that after birth of a vertebrate there is a requirement for the metabolism of Vitamin D₃ (cholecalciferol) to 1,25-(OH)₂-Vitamin D₃ to produce the hormonally active form essential for calcium homeostasis. However it is not known whether the enzymatic capability to produce 1,25-(OH)₂-D₃ only appears after birth or whether it is generated in the embryo. Presented in this paper are results of studies designed to measure the production and localization of 1,25-(OH)₂-D₃ in the embryo. It was found that the renal enzyme, 25-OH-cholecalciferol-1-hydroxylase, which is capable of producing 1,25-(OH)₂-D₃, is present as early as day 9 of incubation (12 days before hatch) in White Leghorn chicks. Further, the enzyme activity increases 6-fold to a maximal level which occurs on the day of hatching. 1,25-(OH)₂-D₃ was shown to be produced in vivo at day 17 and was found then in low levels in the embryonic intestine and kidney. Thus we have shown that 1,25-(OH)₂-D₃ is made by embryonic chick kidneys and is found in low levels in embryonic chick intestine and kidney significantly before hatch.     

Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: A phylogenetic appraisal

We measured the serum concentration of 25-hydroxyvitamin D₃ (25-OH-D₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂-D₃) in 23 different Platyrrhines from four different genera and in 21 Catarrhines from six different genera in residence at the Los Angeles Zoo. The mean (±S.E.) serum concentration of 1,25-(OH)₂-D₃ was significantly greater in Platyrrhines (810 ± 119 pg/ml) than in Catarrhines (61 ± 5 pg/ml), suggesting that high circulating concentrations of the active vitamin D hormone were a characteristic of New World primates in both the Cebidae and Callitrichidae family. This increase in the serum concentration of 1,25-(OH)₂-D₃ is probably an adaptational response on the part of Platyrrhini to offset a relative decrease in the concentration of specific receptor for 1,25-(OH)₂-D₃ in target tissues for the hormone.     

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions

Summary 1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂-D₃) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)₂-D₃ using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)₂-D₃. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)₂-D₃. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)₂-D₃. The antiproliferative effect of 1,25-(OH)₂-D₃ was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)₂-D₃ is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)₂-D₃ to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)₂-D₃, but can be used to assay other agents that modulate keratinocyte proliferation. Portions of this work were presented and abstracted at the April 1988 meeting of the Society of Investigative Dermatology (J. Inv. Derm. 90(4): 586; 1988) and the February 1988 meeting of New York Academy of Sciences (Ann NY Acad. Sci. 548: 341–342; 1988).     

Immunoreactive parathyroid hormone levels in platyrrhini and catarrhini: A comparative analysis with three different assays

Serum concentrations of the hormonal form of vitamin D₃—1,25-dihydroxy-vitamin D₃ [1,25-(OH)₂-D₃]—are elevated in many genera of platyrrhines when compared to catarrhines; this elevation is presumed to result from a decrease in the ability of the target cell receptor effectively to recognize 1,25-(OH)₂-D₃. The activity of the renal 25-hydroxyvitumin D₃-1α-hydroxylase, the mammalian enzyme which synthesizes the majority of the circulating 1,25-(OH)₂-D₃, is accelerated by parathyroid hormone (PTH). In order to determine whether the elevated serum concentrations of 1,25-(OH)₂-D₃ in platyrrhines were the result of relative hyperparathyroidism, we measured serum levels of immunoreactive parathyroid hormone (iPTH) in normocalcemic platyrrhines, catarrhines, and human subjects with assays that recognize different domains of the human PTH molecule. Antisera directed against the biologically active, aminoterminus of PTH yielded comparable mean values for iPTH among three test groups. The mean concentration of iPTH as assessed by a “proximal” midregion assay was significantly reduced in platyrrhine serum when compared to either human or catarrhine serum. A “distal” midregion assay yielded a reduced mean value for iPTH in both platyrrhine and catarrhine serum when compared to human serum. These data suggest that 1) high circulating levels of 1,25-(OH)₂-D₃ in New World primates are not the result of hyperparathyroidism; and 2) structural homology between human and primate PTH diminishes progressively as one moves toward the carboxyterminus of the molecule and is lost more rapidly in the platyrrhine than in the catarrhine hormone.     

Interactions between oestrogen and 1α,25(OH)<Subscript>2</Subscript>-vitamin D<Subscript>3</Subscript> signalling and their roles in spermatogenesis and spermatozoa functions

Oestrogens and 1α,25(OH)₂-vitamin D₃ (1,25-D₃) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D₃ are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D₃ in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.     

Quercetin exerts an inhibitory effect on cellular bioenergetics of the B164A5 murine melanoma cell line

This study examined the hypothesis that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) upregulates the insulin-independent signaling cascade of glucose metabolism. C2C12 myotubes were treated with high glucose (HG, 25 mM) and 1,25(OH)₂D₃ (0–50 nM). 1,25(OH)₂D₃ supplementation upregulated both insulin-independent (SIRT1) and insulin-dependent (p-IRS) signaling molecules, and stimulated the GLUT4 translocation, and glucose uptake in HG-treated myotubes. The effect of 1,25(OH)₂D₃ on IRS1 phosphorylation, GLUT4 translocation, and glucose uptake was attenuated in SIRT1-knockdown myotubes. Treatment with 1,25(OH)₂D₃, coupled with insulin, enhanced GLUT4 translocation and glucose uptake compared to treatment with either insulin or 1,25(OH)₂D₃ alone in HG-treated myotubes, which suggests that insulin-independent signaling molecules can contribute to the higher glucose metabolism observed in 1,25(OH)₂D₃ and insulin-treated cells. The data, therefore, suggest that 1,25(OH)₂D₃ increases glucose consumption by inducing SIRT1 activation, which in turn increases IRS1 phosphorylation and GLUT4 translocation in myotubes.     

The Candidate Oncogene CYP24A1: A Potential Biomarker for Colorectal Tumorigenesis

The main autocrine/paracrine role of the active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1,25-D₃), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D₃ 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D₃, we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D₃ availability, decreasing its antiproliferative effect. (J Histochem Cytochem 58:277–285, 2010)     

Vitamin D Up-regulates Glucose Transporter 4 (GLUT4) Translocation and Glucose Utilization Mediated by Cystathionine-γ-lyase (CSE) Activation and H2S Formation in 3T3L1 Adipocytes

A scientific explanation for the beneficial role of vitamin D supplementation in the lowering of glycemia in diabetes remains to be determined. This study examined the biochemical mechanism by which vitamin D supplementation regulates glucose metabolism in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mm) in the presence or absence of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (25, 50 nm), the active form of vitamin D. 1,25(OH)₂D₃ treatment caused significant up-regulation of GLUT4 total protein expression and its translocation to cell surface, and an increase in glucose uptake as well as glucose utilization in HG-treated cells. 1,25(OH)₂D₃ also caused cystathionine-γ-lyase (CSE) activation and H₂S formation in HG-treated adipocytes. The effect of 1,25(OH)₂D₃ on GLUT4 translocation, glucose utilization, and H₂S formation was prevented by propargylglycine, an inhibitor of CSE that catalyzes H₂S formation. Studies using antisense CSE also demonstrated the inhibition of GLUT4 translocation as well as glucose uptake and utilization in 1,25(OH)₂D₃-supplemented CSE-siRNA-transfected adipocytes compared with controls. 1,25(OH)₂D₃ treatment along with insulin enhanced GLUT4 translocation and glucose utilization compared with either insulin or 1,25(OH)₂D₃ alone in HG-treated adipocytes. 1,25(OH)₂D₃ supplementation also inhibited monocyte chemoattractant protein-1 and stimulated adiponectin secretion in HG-treated adipocytes, and this positive effect was prevented in propargylglycine-treated or CSE-knockdown adipocytes. This is the first report to demonstrate that 1,25(OH)₂D₃ up-regulates GLUT4 translocation and glucose utilization and decreases inflammatory markers, which is mediated by CSE activation and H₂S formation in adipocytes. This study provides evidence for a novel molecular mechanism by which 1,25(OH)₂D₃ can up-regulate the GLUT4 translocation essential for maintenance of glucose metabolism.     

1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

New ionic targets of 3,3′,5′-triiodothyronine at the plasma membrane of rat Sertoli cells

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT₃ on calcium (⁴⁵Ca²⁺) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT₃. Our results indicate that rT₃ induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT₃ occurred in <5 min in Sertoli cells. In addition, the rT₃ metabolite, T₂, also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT₃ also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na⁺/K⁺ ATPase pump. The rapid response of rT₃ is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells.     

Rat testicular germ cells and sertoli cells release different types of bioactive transforming growth factor beta in vitro

Several in vivo studies have reported the presence of immunoreactive transforming growth factor-β's (TGF-β's) in testicular cells at defined stages of their differentiation. The most pronounced changes in TGF-β₁ and TGF-β₂ immunoreactivity occurred during spermatogenesis. In the present study we have investigated whether germ cells and Sertoli cells are able to secrete bioactive TGF-β's in vitro, using the CCl64 mink lung epithelial cell line as bioassay for the measurement of TGF-β. In cellular lysates, TGF-β bioactivity was only observed following heat-treatment, indicating that within these cells TGF-β is present in a latent form. To our surprise, active TGF-β could be detected in the culture supernatant of germ cells and Sertoli cells without prior heat-treatment. This suggests that these cells not only produce and release TGF-β in a latent form, but that they also release a factor which can convert latent TGF-β into its active form. Following heat-activation of these culture supernatant's, total TGF-β bioactivity increased 6- to 9-fold. Spermatocytes are the cell type that releases most bioactive TGF-β during a 24 h culture period, although round and elongated spermatids and Sertoli cells also secrete significant amounts of TGF-β. The biological activity of TGF-β could be inhibited by neutralizing antibodies against TGF-β₁ (spermatocytes and round spermatids) and TGF-β₂ (round and elongating spermatids). TGF-β activity in the Sertoli cell culture supernatant was inhibited slightly by either the TGF-β₁ and TGF-β₂ neutralizing antibody.These in vitro data suggest that germ cells and Sertoli cells release latent TGF-β's. Following secretion, the TGF-β's are converted to a biological active form that can interact with specific TGF-β receptors. These results strengthen the hypothesis that TGF-β's may play a physiological role in germ cell proliferation/differentiation and Sertoli cell function.     

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.     

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50?h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100?nM E2 (17β-oestradiol) or 100?nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2?pmol/cell) at similar rates during the 50?h. After 25?h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50?h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50?h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25?h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.