Tommi Manninen

Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation.

Hearing loss is a major public health problem, and its treatment with traditional therapy strategies is often unsuccessful due to limited drug access deep in the temporal bone. Multifunctional nanoparticles that are targeted to specified cell populations, biodegradable, traceable in vivo, and equipped with controlled drug/gene release may resolve this problem. We developed lipid core nanocapsules (LNCs) with sizes below 50 nm. The aim of the present study is to evaluate the ability of the LNCs to pass through the round window membrane and reach inner ear targets. FITC was incorporated as a tag for the LNCs and Nile Red was encapsulated inside the oily core to assess the integrity of the LNCs. The capability of LNCs to pass through the round window membrane and the distribution of the LNCs inside the inner ear were evaluated in rats via confocal microscopy in combination with image analysis using ImageJ. After round window membrane administration, LNCs reached the spiral ganglion cells, nerve fibers, and spiral ligament fibrocytes within 30 min. The paracellular pathway was the main approach for LNC penetration of the round window membrane. LNCs can also reach the vestibule, middle ear mucosa, and the adjacent artery. Nuclear localization was detected in the spiral ganglion, though infrequently. These results suggest that LNCs are potential vectors for drug delivery into the spiral ganglion cells, nerve fibers, hair cells, and spiral ligament.

Vitamin D and prostate cancer.

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(2000) (847-852)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had 25OH-vitamin D (25-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum 25-VD, whereas low serum 25-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest 25-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high 25-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum 25-VD concentrations. After 1-month exposure, there was no significant increase in the serum 25-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that 25-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and 24-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-2, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since keratinocyte growth factor (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

Role of 24-hydroxylase in vitamin D3 growth response of OVCAR-3 ovarian cancer cells

Vitamin D and its analogues are potent regulators of cell growth and differentiation both in vivo and in vitro. We studied the effects of 25‐hydroxyvitamin D3 [25(OH)D3], 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] and vitamin D analogue, EB 1089, on the growth of a human ovarian cancer cell line, OVCAR‐3. We also studied the expression of vitamin D metabolising enzymes 24‐hydroxylase (24OHase) and 1α‐hydroxylase (1αOHase). Our results showed that high concentrations (10 and 100 nM) of 1,25(OH)2D3 inhibited a cell proliferation, whereas low concentration (0.1 nM) stimulated growth of the OVCAR‐3 cells. In the concentration range of 10–500 nM a prohormone, 25(OH)D3, stimulated growth. An amount of 1 nM EB 1089 and 100 nM 1,25(OH)2D3 inhibited growth with an equal magnitude. The expression of 24OHase was strongly induced by 1,25(OH)2D3 and EB 1089 in OVCAR‐3 cells, and analysis of vitamin D metabolites showed the functionality of 24OHase. An inhibition of 24OHase activity with a novel 24OHase inhibitor enhanced growth‐inhibiting effects of 1,25(OH)2D3 and suppressed the growth stimulation of 100 nM 25(OH)D3. We also report the expression of a vitamin D activating enzyme, 1αOHase, in 7 ovarian cancer cell lines. The production of 1,25(OH)2D3 in OVCAR‐3 cells was low, possibly due to an extensive activity of 24OHase or a low 1αOHase activity. These results suggest that in ovarian cancer cells vitamin D metabolizing enzymes might play a key role in modulating the growth response to vitamin D. The possible mitogenic effects of vitamin D should be considered when evaluating treatment of ovarian cancer with vitamin D.

Heat shock protein 90 and the nuclear transport of progesterone receptor.

Abstract Steroid receptors exist as large oligomeric complexes in hypotonic cell extracts. In the present work, we studied the nuclear transport of the 2 major components of the oligomeric complex, the receptor itself and the heat shock protein 90 (Hsp90), by using different in vitro transport systems: digitonin permeabilized cells and purified nuclei. We demonstrate that the stabilized oligomeric complex of progesterone receptor (PR) cannot be transported into the nucleus and that unliganded PR salt dissociated from Hsp90 is transported into the nucleus. When nonstabilized PR oligomer was introduced into the nuclear transport system, the complex dissociated and the PR but not the Hsp90 was transported into the nucleus. If PR exists as an oligomeric form after synthesis, as suggested by the experiments with reticulocyte lysate, the present results suggest that the complex is short-lived and is dissociated before or during nuclear transport. Thus, the role of Hsp90 in PR action is likely to reside in the Hsp90-assisted chaperoning process of PR preceding nuclear transport of the receptor.

Gene expression profiles in human and mouse primary cells provide new insights into the differential actions of vitamin D3 metabolites.

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1 −/−), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1 −/−. By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.

Interaction of nuclear receptors with hsp90 in living cells

The ubiquitous heat shock protein 90 (hsp90) has been shown to participate directly in the function of a wide variety of cellular signal transduction components, including steroid receptors (SRs). However, there is still no direct evidence for an in vivo association of SRs with hsp90. This study utilizes the mammalian two-hybrid system to study the ability of hsp90 to interact with various (non)liganded nuclear receptors (NRs) in vivo in mammalian cells. As bait, we used ligand-binding domain (LBD) of various NRs fused with the GAL4-DBD. hsp90/Receptor interactions were monitored in COS cells. When NR-LBDs were co-transfected along with hsp90/VP16, none (RxR(2)-LBD) or only minimal (SR-LBDs) transcription inductions were observed (1.9-4.7-fold) in the absence of ligand. Addition of ligand further abolished the observed minimal induction. As a positive control for interaction we used TIF-2, which interacts with SRs in a ligand inducible manner. When co-transfected with NR-LBDs in the absence of ligand TIF-2/VP16 induced minimal activation of transcription (1.6-4.5-fold) that was comparable to the activation induced by the NR-LBDs. In contrast, in the presence of the ligand, the activation ranged between 62- and 134-fold depending on the receptor. The results suggest that the interaction of SRs with the hsp90 is minimal when compared to a bona fide type of interaction with the co-factors.