Matthew J. Beckman

Modulation of nerve growth factor in peripheral organs by estrogen and progesterone.

Nerve growth factor (NGF) synthesized in peripheral organs plays a critical role in the development and maintenance of the nervous system and also participates in processing nociceptive stimuli. Previous studies suggest that reproductive hormones may regulate the expression of NGF. Ovariectomies were performed on female mice, and mice were killed 24 h after hormone replacement to evaluate the effects of estrogen and progesterone on NGF in peripheral organs, specifically the uterus, bladder, heart, and salivary gland. Sham-operated intact mice and untreated ovariectomized mice served as controls. Immunohistochemistry demonstrated the presence of NGF, estrogen receptor-alpha, estrogen receptor-beta, and progesterone receptors in these organs. Ovariectomy caused a significant decrease in NGF protein content in the uterus, and short term treatment of ovariectomized mice with estrogen and/or progesterone increased uterine NGF mRNA and restored NGF protein to concentrations similar to intact control mice. Ovariectomy did not affect NGF protein concentrations in the salivary gland, but treatment of ovariectomized mice with estrogen alone or in conjunction with progesterone stimulated concentrations of NGF protein that exceeded those observed in intact control or ovariectomized, untreated mice. NGF mRNA was increased in salivary glands from ovariectomized mice treated with progesterone alone or in combination with estrogen relative to other groups. NGF protein content of the hearts of ovariectomized mice treated with estrogen alone or in conjunction with progesterone was increased relative to intact controls and ovariectomized, untreated mice, but neither ovariectomy or hormone replacement affected NGF mRNA content in the heart. NGF protein content of the bladder was unaffected by ovariectomy or hormone treatment, and bladder NGF mRNA was unaffected by ovariectomy or hormone treatment. Collectively, these results indicate that reproductive hormones have the capacity to regulate NGF message and protein in a manner that varies among organs. Fluctuations in the expression of NGF, in conjunction with other factors, may help to explain gender differences in pain sensation and inflammatory response.

Dual functions of autophagy in the response of breast tumor cells to radiation: Cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D3

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.

Multiple factor interactions in biomimetic mineralization of electrospun scaffolds

One of the major limitations in scaffold-based bone tissue engineering has been the inability to increase the loading of biologically active inorganic mineral. The present study introduces a novel two step strategy to increase overall mineral content of electrospun scaffolds and employs multiple factor interaction as a statistic to identify the combination of factors that yields maximal scaffold mineralization. Different amounts of nHA (0, 10, 25 and 50% by wt. of polymer) were electrospun in combination with polydioxanone (PDO) or poly(glycolide: lactide) to generate composite scaffolds. Successful incorporation of nHA within, on and in between nanofibers was confirmed by transmission and scanning electron microscopy. These scaffolds were immersed in different types (conventional, revised, ionic and modified) of simulated body fluid (SBF), prepared at 1x and 4x concentrations and the incubation was carried out either in static or dynamic setting at biomimetic conditions. At 2 weeks, the total amount of mineral within the scaffold was quantified using a modified Alizarin Red-based assay. Each of the five independent factors was analyzed independently and tested for interaction using random effects ANOVA. Statistics revealed significant higher order interactions among factors and the combination of PDO containing 50% nHA incubated in 1x revised SBF resulted in maximum mineralization.

A Switch Between Cytoprotective and Cytotoxic Autophagy in the Radiosensitization of Breast Tumor Cells by Chloroquine and Vitamin D

Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution, and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer, and demonstrate the existence of an “autophagic switch” from cytoprotective autophagy with radiation alone to cytotoxic autophagy with the 1,25 D3–radiation combination.

Extracellular calcium is a direct effecter of VDR levels in proximal tubule epithelial cells that counter-balances effects of PTH on renal Vitamin D metabolism

In renal proximal tubules, VDR is transiently decreased by parathyroid hormone (PTH) during times of hypocalcemia and returns to normal levels with the rise in serum calcium (Ca). In this study we tested the hypothesis that elevated extracellular Ca induces VDR in a human renal proximal cell line (HK-2G) stably expressing PTH receptor type I. Exposure of HK-2G cells to increasing Ca concentration, up to 3mM, induced the expression of VDR. The increase in VDR occurred within 1h and was sustained over 24h. The increase in VDR was also dose-dependently increased using 20-100 nM gadolinium, suggesting the induction of VDR is regulated via the extracellular Ca sensing receptor (CaSR) with is naturally expressed in HK-2G cells. In conclusion, an extracellular Ca concentration in the physiological range is capable of direct increase of renal proximal VDR expression, and the induction mechanism represents a strategy the body may use to counterbalance effects of PTH on renal Vitamin D metabolism.

Multipotent adipose stromal cells and breast cancer development: Think globally, act locally.

It has long been appreciated that stromal cells within the breast tumor microenvironment contribute to mammary carcinogenesis. However, to date, very little is known regarding the role of local adipose‐derived stromal cells (ASCs) in the development of breast cancer. Based on pathological, epidemiological and experimental data, we postulate that breast‐derived ASCs are unique mesenchymal stem‐like cells that play a critical role in the development of breast cancer and discuss the global implications of this working model in terms of breast cancer prevention, early detection, and new targeted therapies. Mol. Carcinog.

Extracellular Calcium-sensing Receptor Activation Induces Vitamin D Receptor Levels in Proximal Kidney HK-2G Cells by a Mechanism That Requires Phosphorylation of p38α MAPK

In hypocalcaemia, elevated parathyroid hormone transitorily down-regulates the kidney vitamin D receptor, which returns to normal levels with the rise in serum extracellular calcium [Ca2+]e. In this study, we investigated the mechanism that underlies VDR increase in kidney in association with elevated [Ca2+]e. Examination of MAP kinase signals in a proximal tubule human kidney (HK-2G) epithelial cell line showed that treatment of [Ca2+]e in the culture medium elevated phosphorylation of both ERK and p38 MAPKs. Blockade of p38 phosphorylation with SB203580 or SB202190 in turn abolished [Ca2+]e-mediated VDR protein increase, while treatment with PD98059 and U0126, specifically blocked ERK phosphorylation, but had no effect on VDR stimulation by [Ca2+]e. Furthermore, SB203580 treatment potently repressed [Ca2+]e-mediated activation of VDR promoter. We also demonstrate that si-RNA knock down of p38α completely diminished high [Ca2+]e-mediated VDR induction. Direct CaSR involvement was demonstrated by using an si-RNA of CaSR that impeded [Ca2+]e-mediated induction of VDR. In conclusion, a high extracellular [Ca2+]e concentration in the physiological range is capable of directly increasing renal proximal VDR expression, and the induction mechanism requires activation of the CaSR and signal mediation by the p38α MAP kinase pathway.

Mesenchymal stem cells in mammary adipose tissue stimulate progression of breast cancer resembling the basal-type

Data are accumulating to support a role for adipose-derived mesenchymal stem cells (MSCs) in breast cancer progression; however, to date most studies have relied on adipose MSCs from non-breast sources. There is a particular need to investigate the role of adipose MSCs in the pathogenesis of basal-like breast cancer, which develops at a disproportionate rate in pre-menopausal African-American women with a gain in adiposity. The aim of this study was to better understand how breast adipose MSCs (bMSCs) contribute to the progression of basal-like breast cancers by relying on isogenic HMT-3255 S3 (pre-invasive) and T4-2 (invasive) human cells that upon transplantation into nude mice resemble this tumor subtype. In vitro results suggested that bMSCs may contribute to breast cancer progression in multiple ways. bMSCs readily penetrate extracellular matrix components in part through their expression of matrix metalloproteinases 1 and 3, promote the invasion of T4-2 cells and efficiently chemoattract endothelial cells via a bFGF-independent, VEGF-A-dependent manner. As mixed xenografts, bMSCs stimulated the growth, invasion and desmoplasia of T4-2 tumors, yet these resident stem cells showed no observable effect on the progression of pre-invasive S3 cells. While bMSCs form vessel-like structures within Matrigel both in vitro and in vivo and chemoattract endothelial cells, there appeared to be no difference between T4-2/bMSC mixed xenografts and T4-2 xenografts with regard to intra- or peri-tumoral vascularity. Collectively, our data suggest that bMSCs may contribute to the progression of basal-like breast cancers by stimulating growth and invasion but not vasculogenesis or angiogenesis.

5-HT5a receptors in the carotid body chemoreception pathway of rat.

By using a specific antibody, 5-HT5a receptor-like immunoreactivity was revealed in the chemoreceptive, oxygen sensitive, carotid body (CB) type I cells, and neurons of the petrosal ganglion (PG) and the superior cervical ganglion (SCG) in rat. mRNA encoding for the 5-HTa receptor was also detected in these tissues by RT-PCR, and confirmed with DNA sequencing. The present study provides direct evidence that 5-HT5a receptors are expressed in the CB, PG and SCG, which all likely play fundamental roles in arterial chemoreception.

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression.

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)2D3 concentrations decreased with age in rats of both genders. Abundance of calbindin-D28K and its mRNA in kidney and calbindin-D9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)2D3 may be responsible for low plasma 1,25-(OH)2D3 concentrations observed in older animals. Plasma PTH and 1,25-(OH)2D3 concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D9K and calbindin-D9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)2D3 concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)2D3-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.