Ilona Mandrika

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

Human OX1 orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB1 cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX1, OX2 and CB1 receptors, C-terminally fused with either Renilla luciferase or GFP(2) green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB1 receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP(2) to CB1 produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX1-OX2 interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB1 receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.

Evidence for constitutive dimerization of niacin receptor subtypes.

The recently deorphanized niacin receptor subtypes NIACR1 (GPR109A) and NIACR2 (GPR109B) play an essential role in the regulation of metabolic processes and immune reactions. Both receptors belong to the G-protein-coupled receptor (GPCR) family, whose members have traditionally been treated as monomeric entities, but now appear to exist and function as both homodimers and heterodimers. In this study, a close physical interaction is shown between the highly homologous niacin receptor subtypes, NIACR1 and NIACR2, using bioluminescence resonance energy transfer (BRET(2)) in living cells. The extent of homo- and hetero-dimerization of the niacin receptors did not vary after activation of the receptors with selective agonists, indicating that the dimerization state of NIACR1 and NIACR2 is not regulated by ligand binding. Moreover, detection of niacin receptor dimers in both plasma membrane- and endoplasmic reticulum-enriched fractions suggests that they are formed early in the biosynthetic pathway. Taken together, these results demonstrate that niacin receptor dimerization is a constitutive process occurring early during biosynthesis.

Melanoma risk associated with MC1R gene variants in Latvia and the functional analysis of rare variants

To evaluate the association of melanocortin 1 receptor gene (MC1R) variants with melanoma risk in a Latvian population, the MC1R gene was sequenced in 200 melanoma patients and 200 control persons. A functional study of previously uncharacterized, rare MC1R variants was also performed. In total, 26 different MC1R variants, including two novel variants Val165Ile and Val188Ile, were detected. The highest risk of melanoma was associated with the Arg151Cys variant (odds ratio (OR) 4.47, 95% confidence interval (CI) 2.19-9.14, P<0.001). A gene dosage effect was observed, with melanoma risk for carriers of two variants being twice (OR 3.98, 95% CI 2.15-7.38, P<0.001) that of carriers of one variant (OR 1.98, 95% CI 1.26-3.11, P=0.003). After stratification according to the pigmentation phenotype, the risk of melanoma remained in groups with otherwise protective phenotypes. Functional analyses of eight previously uncharacterized MC1R variants revealed that a subset of them is functionally relevant. Our results support the contribution of MC1R variants to a genetic predisposition to melanoma in Latvia.

Functional Characteristics of Multipotent Mesenchymal Stromal Cells from Pituitary Adenomas

Pituitary adenomas are one of the most common endocrine and intracranial neoplasms. Although they are theoretically monoclonal in origin, several studies have shown that they contain different multipotent cell types that are thought to play an important role in tumor initiation, maintenance, and recurrence after therapy. In the present study, we isolated and characterized cell populations from seven pituitary somatotroph, nonhormonal, and lactotroph adenomas. The obtained cells showed characteristics of multipotent mesenchymal stromal cells as observed by cell morphology, cell surface marker CD90, CD105, CD44, and vimentin expression, as well as differentiation to osteogenic and adipogenic lineages. They are capable of growth and passaging under standard laboratory cell culture conditions and do not manifest any hormonal cell characteristics. Multipotent mesenchymal stromal cells are present in pituitary adenomas regardless of their clinical manifestation and show no considerable expression of somatostatin 1–5 and dopamine 2 receptors. Most likely obtained cells are a part of tissue-supportive cells in pituitary adenoma microenvironment.

BRCA1 gene-related hereditary susceptibility to breast and ovarian cancer in Latvia

PURPOSE In this report, we summarise data on BRCA1 gene analysis in Latvia to characterise criteria of genetic testing for breast and ovarian cancer susceptibility. MATERIAL/METHODS Analysis by SSCP/HD, MALDI-TOF mass spectrometry or DNA sequencing was used for mutation detection. Mutations identified were confirmed by direct DNA sequencing. RESULTS Out of 1068 breast and 231 ovarian cancer patients from different families: 58 carried the c.5266dupC and 43 carried the c.4035delA mutations. Every 4th patient in our study did not report cancer in the family. The breast cancer was diagnosed earlier in carriers of the c.5266dupC than in carriers of the c.4035delA (p=0.003). The incidence of breast or ovarian cancer does not differ among the 2 mutation carriers in our patient group. The nature of the c.5266dupC mutation might be more deleterious. CONCLUSIONS We recommend the screening of 4 founder BRCA1 mutations in all breast and ovarian cancer patients in Latvia at diagnosis of disease regardless of family history or age. The BRCA1 screening can be carried out efficiently using the MALDI-TOF mass spectrometry mutation detection method developed in the Biomedical Research and Study Centre (Riga, Latvia).

Hydroxycarboxylic Acid Receptor Ligands Modulate Proinflammatory Cytokine Expression in Human Macrophages and Adipocytes without Affecting Adipose Differentiation

Members of the hydroxycarboxylic acid receptor (HCA1-3) family are mainly expressed in adipocytes and immune cells. HCA2 ligand, niacin, has been used for decades as lipid-modifying drug. Recent studies suggest that HCA ligands can be involved in the modulation of inflammatory processes. In this study, we evaluated the effects of HCA1-3 ligands on adipose differentiation and cytokine expression in human adipocytes and macrophages. Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes were induced to differentiate into adipocytes for 8 d in the presence or absence of HCA ligands and evaluated for lipid accumulation and adipogenic gene expression. The inhibitory effects of the ligands on the expression and production of cytokines were measured in lipopolysaccharide (LPS)-stimulated adipocytes and THP-1 macrophage cells. Preadipocytes treated with HCA ligands showed no changes in the capacity to differentiate into adipocytes and no significant alteration in peroxisome proliferator activated receptor γ (PPARγ) or its target gene expression. HCA2-3 ligands significantly downregulated LPS-induced expression of interleukin (IL)-6 (53-64%), tumor necrosis factor-α (TNF-α) (55-69%) and IL-8 (51-59%) in adipocytes and macrophages. IL-1β inhibition (58-68%) by HCA2-3 ligands was observed only in adipocytes. Furthermore, LPS increased the expression of HCA2-3 in adipocytes and macrophages and this expression was decreased by treatment with their ligands. These results suggest that HCA ligands modulated LPS-mediated pro-inflammatory gene expression in both macrophages and adipocytes without affecting adipogenesis. Therefore, targeting HCA2 and HCA3 would be beneficial in treating inflammation conditions associated with atherosclerosis and obesity.

Synthesis and evaluation of ( E )-2-(5-phenylpent-2-en-4-ynamido)cyclohex-1-ene-1-carboxylate derivatives as HCA2 receptor agonists

Novel series of compounds consisting of 2-amidocyclohex-1-ene carboxylate and phenyl parts which are connected by enyne (compounds 2a-f), but-1-yne (compounds 4a-j), and phenylethylene (compounds 5a-f) linkers as HCA2 full agonists were designed and their functional activity using cAMP assay and binding affinity using radioligand (3H-niacin) binding assay were evaluated. In general, compounds of all three series exhibit similar HCA2 binding and activation profile. However, the activity is strongly dependent on the substituent at the aromatic part of the structure. Among the structures evaluated, the highest affinity and potency in all series were exhibited by compounds containing 4-hydroxy and/or 2-chloro or 2-fluoro substituents. The most active compounds in the enyne and but-1-yne series in the cAMP assay are 2-fluoro,4-hydroxy and 2-chloro,4-hydroxy phenyl derivatives 2f, 4f, and 4g showing potency similar to the previously described 4-hydroxy-biphenyl analogue 5c.