Timo Ylikomi

Characterisation of human dental stem cells and buccal mucosa fibroblasts

Human craniofacial stem cells are recently discovered sources of putative mesenchymal stem cells that hold great promise for autogenic or allogenic cell therapy and tissue engineering. Prior to employing these cells in clinical applications, they must be thoroughly investigated and characterized. In this study, the surface marker expression was investigated on dental pulp stem cells (DPSCs), dental follicle cells (DFCs), periodontal ligament stem cells (PDLSCs), and buccal mucosa fibroblasts (BMFs) utilising surface markers for flow cytometry. The osteogenic potential was also examined by bone-associated markers alkaline phosphatase, Runx2, collagen type I, osteocalcin, and osteopontin. The results from our study demonstrate that the dental cell sources exhibit comparable surface marker and bone-associated marker profiles parallel to those of other mesenchymal stem cell sources, yet distinct from the buccal mucosa fibroblasts. Our data support evidence towards clinical applicability of dental stem cells in hard tissue regeneration.

Association between serum 25(OH)D concentrations and bone stress fractures in Finnish young men.

Low vitamin D level may predict rickets, osteomalacia, or osteoporosis. We examined serum 25(OH)D concentration as a predisposing factor for bone stress fracture in 756 military recruits. The average serum 25(OH)D concentration was significantly lower in the group with fracture, suggesting a relationship between vitamin D and fatigue bone stress fracture.

Vitamin D supplementation for the prevention of acute respiratory tract infection: a randomized, double-blinded trial among young Finnish men.

Ilkka Laaksi, Juha-Petri Ruohola, Ville Mattila, Anssi Auvinen, Timo Ylikomi, and Harri Pihlajamaki Department of Cell Biology, Medical School, and Department of Epidemiology, Tampere School of Public Health, University of Tampere, and Department of Clinical Chemistry, Tampere University Hospital, Tampere, and Finnish Defence Forces and Research Department, Centre for Military Medicine, Helsinki, Finland

Antiproliferative Action of Vitamin D

During the past few years, it has become apparent that vitamin D may play an important role in malignant transformation. Epidemiological studies suggest that low vitamin D serum concentration increases especially the risk of hormone-related cancers. Experimentally, vitamin D suppresses the proliferation of normal and malignant cells and induces differentiation and apoptosis. In the present review we discuss the mechanisms whereby vitamin D regulates cell proliferation and whether it could be used in prevention and treatment of hyperproliferative disorders like cancers.

25-Hydroxyvitamin D3 is an active hormone in human primary prostatic stromal cells

According to the present paradigm, 1α,25‐dihydroxyvitamin D3 [1α,25‐(OH)2D3] is a biologically active hormone; whereas 25‐hydroxyvitamin D3 (25OHD3) is regarded as a prohormone activated through the action of 25‐hydroxyvitamin D3 1α‐hydroxylase (1α‐ hydroxylase). Although the role of vitamin D3 in the regulation of growth and differentiation of prostatic epithelial cells has been well studied, its action and metabolism in prostatic stroma are still largely unknown. We investigated the effects of 25OHD3 and 1α,25‐(OH)2D3 on two human stromal primary cultures termed P29SN and P32S. In a cell proliferation assay, 25OHD3 was found at physiological concentrations of 100–250 nM to inhibit the growth of both primary cultures, whereas 1α,25‐(OH)2D3 at a pharmacological concentration of 10 nM exhibited the growth‐inhibitory effects on P29SN cells but not on P32S cells. Quantitative real‐time RT‐PCR analysis revealed that both 25OHD3 and 1α,25‐(OH)2D3 induced 25‐hydroxyvitamin D3 24‐ hydroxylase (24‐hydroxylase) mRNA in a dose‐ and time‐dependent manner. By inhibiting 1α‐ hydroxylase and/or 24‐hydroxylase enzyme activities, the induction of 24‐hydroxylase mRNA by 250 nM 25OHD3 was clearly enhanced, suggesting that 1α‐hydroxylation is not a prerequisite for the hormonal activity of 25OHD3. Altogether our results suggest that 25OHD3 at a high but physiological concentration acts as an active hormone with respect to vitamin D3 responsive gene regulation and suppression of cell proliferation.

Vitamin D inhibits myometrial and leiomyoma cell proliferation in vitro.

OBJECTIVE To determine the effect of 1,25(OH)(2)D(3) and 25(OH)D(3) vitamin D derivates on the growth of leiomyoma and myometrial cells in vitro. DESIGN In vitro study. SETTING Cell biology research laboratory. PATIENT(S) Six premenopausal women with uterine leiomyomas undergoing hysterectomy. INTERVENTION(S) Samples of leiomyomas and normal myometrial tissue were obtained, and paired cultures were established. MAIN OUTCOME MEASURE(S) A colorimetric crystal violet assay to determine the effect of 1,25(OH)(2)D(3) and 25(OH)D(3) on cell growth. RESULT(S) In both myometrial and leiomyoma cells, 0.1 nM physiologic level of 1,25(OH)(2)D(3) inhibited growth by 12% when compared with controls. The growth inhibition was concentration dependent; the highest concentration of 1,25(OH)(2)D(3) (100 nM) inhibited growth by 62% in both cell types. All the differences were statistically significant. A slight stimulation (<4%) of cell proliferation was observed with the lowest 25(OH)(2)D(3) concentrations. When treated with either a 500 nM or 1000 nM concentration of the compound, the growth of both cell types fell to approximately 50% of that of the control cultures, and the level of inhibition with the latter concentration was statistically significant. CONCLUSION(S) Both myometrial and leiomyoma cell growth in vitro was effectively inhibited by 1,25(OH)(2)D(3). Vitamin D may play a role in the growth of uterine leiomyomas.

Expression of Nuclear Receptors and Cofacotrs in Human Endometrium and Myometrium

Objective: To study the expression of nuclear receptors and cofactors in human endometrium and myometrium in proliferative and secretory phases of the menstrual cycle. Methods: Multiprobe ribonuclease protection assay and real-time reverse transcriptase polymerase chain reaction were used to quantitate mRNA levels of steroid receptors, vitamin D receptor (VDR), retinoic acid receptors (RAR), and cofactors AIB1 (amplified in breast cancer-1), CBP (cyclic adenosine monophosphate response element binding protein), pCAF (p300/CBP-associated factor), TIF2 (transcription intermediary factor-2), N-CoR (nuclear receptor corepressor), and SMRT (silencing mediator of repressed transcription). Cyclin A expression was analyzed to determine the proliferation status of the tissues. Results: The expression of androgen receptor, estrogen receptors α and β, progesterone receptor, and RARα followed cyclin A expression. There was more abundant expression in the proliferative phase endometrium than in the secretory phase endometrium. Glucocorticoid receptor, VDR, RARβ, and RARγ were stably expressed during the menstrual cycle in both endometrium and myometrium. Cofactors N-CoR, SMRT, pCAF, CBP, TIF2, AIB1, and p300 mRNAs were expressed in all samples in both endometrium and myometrium. N-CoR, pCAF, AIB1, and p300 appeared not to be regulated when comparing proliferative and secretory phases of the cycle. Individual differences were found in the expression levels of both nuclear receptors and cofactors. Conclusion: The menstrual cycle-dependent regulation of nuclear receptor expression was more apparent in the endometrium that in the myometrium, whereas cofactor expression was cycle dependent. There were individual differences in the expression levels of different receptors and cofactors. In hormonal therapy these differences might result in different responses, depending on the patient as well as the ligand used.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

Abstract A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

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.

EVIDENCE FOR ENHANCED UBIQUITIN-MEDIATED PROTEOLYSIS OF THE CHICKEN PROGESTERONE RECEPTOR BY PROGESTERONE

Genomic actions of progesterone are mediated via A and B isoforms of the progesterone receptor (PR). One major factor controlling PR level is progesterone causing negative autoregulation (down-regulation) of the receptor protein. In this work we studied the mechanism whereby progesterone exerts its effects on PR level in the chicken oviduct. We found that progesterone does not markedly regulate PR mRNA expression. Furthermore, we demonstrate here for the first time that PR is a target for ubiquitylation and that the proportion of ubiquitylated PR is increased by progesterone treatment. Our data suggest that ligand-induced down-regulation of PR involves enhanced degradation of receptor protein by ubiquitin-proteasome system in vivo.