Roni J. Bollag

Identification of Caveolin and Caveolin-Related Proteins in the Brain

Caveolae are 50–100 nm, nonclathrin-coated, flask-shaped plasma membrane microdomains that have been identified in most mammalian cell types, except lymphocytes and neurons. To date, multiple functions have been ascribed to caveolae, including the compartmentalization of lipid and protein components that function in transmembrane signaling events, biosynthetic transport functions, endocytosis, potocytosis, and transcytosis. Caveolin, a 21–24 kDa integral membrane protein, is the principal structural component of caveolae. We have initiated studies to examine the relationship of detergent-insoluble complexes identified in astrocytes to the caveolin–caveolae compartment detected in cells of peripheral tissues. Immunolocalization studies performed in astrocytes reveal caveolin immunoreactivity in regions that correlate well to the distribution of caveolae and caveolin determined in other cell types, and electron microscopic studies reveal multiple clusters of flask-shaped invaginations aligned along the plasma membrane. Immunoblot analyses demonstrate that detergent-insoluble complexes isolated from astrocytes are composed of caveolin-1α, an identification verified by Northern blot analyses and by the cloning of a cDNA using reverse transcriptase-PCR amplification from total astrocyte RNA. Using a full-length caveolin-1 probe, Northern blot analyses suggest that the expression of caveolin-1 may be regulated during brain development. Immunoblot analyses of detergent-insoluble complexes isolated from cerebral cortex and cerebellum identify two immunoreactive polypeptides with apparent molecular weight and isoelectric points appropriate for caveolin. The identification of caveolae microdomains and caveolin-1 in astrocytes and brain, as well as the apparent regulation of caveolin-1 expression during brain development, identifies a cell compartment not detected previously in brain.

Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

CONTEXT Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). OBJECTIVE The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. METHODS A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. RESULTS We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2. CONCLUSIONS KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production.

Impact of Glucose-Dependent Insulinotropic Peptide on Age-Induced Bone Loss†

GIP is an important hormonal link between nutrition and bone formation. We show for the first time that BMSCs express functional GIP receptors, that expression decreases with aging, and that elevations in GIP can prevent age‐associated bone loss.

Microarray analysis of Tbx2-directed gene expression: A possible role in osteogenesis

Tbx2 is a member of the developmentally important transcriptional regulatory T-box gene family, whose target genes have not been well characterized. In an attempt to identify genes that may be regulated by Tbx2, mouse cDNA microarrays were used to analyze differential gene expression profiles, comparing stably transfected NIH3T3 cells overexpressing Tbx2 and vector-transfected controls. Among 8734 genes, 107 genes were up-regulated by 2-fold or greater, and 66 genes were down-regulated by 2-fold or greater. Caveolin, pleiotrophin (osf-1), osteoblast-specific factor-2 (osf-2) and collagen type I alpha were among the genes upregulated in the Tbx2-overexpressing cells, whereas cadherin 3, tenascin C, and insulin-like growth factor binding protein 10/CYR61 (IBP10) were among the genes downregulated. Northern blot analysis confirmed the correlation of expression of several genes, including IBP10 and osf-2, in fibroblast NIH3T3 and rat osteosarcoma ROS17/2.8 cells differentially expressing Tbx2. In ROS17/2.8 cells transfected with antisense Tbx2, osf-2 was downregulated, whereas transfection of sense Tbx2 upregulated this gene. Interestingly, the expression of pleiotrophin (osf-1) and collagen I alpha with Tbx2 transfection showed an inverse regulatory correlation between NIH3T3 and ROS17/2.8 cells. Thus, Tbx2 can act as both a repressor and activator, and the cellular context can influence the effect on gene expression. Although the data do not address whether Tbx2 directly mediates the transcriptional effect, a number of candidate genes possess putative T-box gene regulatory elements. The results support the hypothesis that Tbx2 may be an important modulator of bone development. Further functional cluster analysis indicates that Tbx2 might also be involved in the regulation of cell cycle and cell adhesion.

Effects of glucose-dependent insulinotropic peptide on behavior

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that rises rapidly in response to nutrient ingestion. The GIP receptor is widely expressed in the brain including the brain stem, telencephalon, diencephalon, olfactory bulb, pituitary, and cerebellum. Until recently it was not clear what the endogenous ligand for this receptor was because no GIP expression had been demonstrated in the brain. GIP synthesis has now been documented in the dentate gyrus of the hippocampus. To define GIP effects on behavior we utilized a mouse model a GIP-overexpressing transgenic mouse (GIP Tg). Specifically, anxiety-related behavior, exploration, memory, and nociception were examined. Compared to age-matched adult male C57BI/6 controls GIP Tg mice displayed enhanced exploratory behavior in the open-field locomotor activity test. GIP Tg mice also demonstrated increased performance in some of the motor function tests. These data suggest that the GIP receptor plays a role in the regulation of locomotor activity and exploration. To our knowledge, this is the first report of effects of GIP on behavior.

Glucose-dependent insulinotropic peptide signaling pathways in endothelial cells

Glucose-dependent insulinotropic peptide (GIP) potentiates glucose-induced insulin secretion. In addition, GIP has vasoconstrictive or vasodilatory properties depending on the vascular bed affected. In order to assess whether this effect could be related to differences in GIP receptor expression, several different endothelial cell types were examined for GIP receptor expression. GIP receptor splice variants were detected and varied depending on the endothelial cell type. Furthermore, stimulation of these cells with GIP led to cell type dependent differences in activation of the calcium and cAMP signaling pathways. To our knowledge this is the first physiological characterization of receptors for GIP in endothelial cells.

1,25-Dihydroxyvitamin D3 Induces Phospholipase D-1 Expression in Primary Mouse Epidermal Keratinocytes

The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) elicits the programmed pattern of differentiation in epidermal keratinocytes. Based on data indicating a potential role of phospholipase D (PLD) in mediating keratinocyte differentiation, we investigated the effect of 1,25(OH)2D3 on PLD expression. A 24-h exposure to 1,25(OH)2D3 stimulated PLD-1, but not PLD-2, mRNA expression. This 1,25(OH)2D3-enhanced expression was accompanied by increased total PLD and PLD-1 activity. Time course studies indicated that 1,25(OH)2D3induced PLD-1 expression by 8 h, with a maximal increase at 20–24 h. Exposure to 1,25(OH)2D3 inhibited proliferation over the same time period with similar kinetics. Expression of the early (spinous) differentiation marker keratin 1 decreased in response to 1,25(OH)2D3 over 12–24 h. Treatment with 1,25(OH)2D3 enhanced the activity of transglutaminase, a late (granular) differentiation marker, by 12 h with a maximal increase after 24 h. In situ hybridization studies demonstrated that the highest levels of PLD-1 expression are in the more differentiated (spinous and granular) layers of the epidermis, with little expression in basal keratinocytes. Our results suggest a role for PLD expression/activity during keratinocyte differentiation.

Glucose-dependent insulinotropic peptide stimulates proliferation and TGF-β release from MG-63 cells

Glucose-dependent insulinotropic peptide (GIP) is known to modulate alkaline phosphatase activity and collagen type I message in osteoblastic-like cells. GIP effects on cell proliferation are not known. We report that GIP dose dependently stimulated 3H-thymidine incorporation in the osteoblastic-like cell line MG-63. Furthermore, GIP increased message and secretion of transforming growth factor beta (TGF-beta), an agent known to regulate osteoblastic proliferation and differentiation. However, when GIP was added to MG-63 cells concurrently with a TGF-beta neutralizing antibody, there was no effect on 3H-thymidine incorporation in these cells. These data demonstrate that GIP stimulates osteoblastic-like cell proliferation but that this effect is not mediated by TGF-beta.

1,25-Dihydroxyvitamin D3, phospholipase D and protein kinase C in keratinocyte differentiation

1,25-Dihydroxyvitamin D(3), thought to be a physiological regulator of epidermal keratinocyte growth and differentiation, also elicits the complete differentiative program in vitro, with expression of various genes/proteins characteristic of both early and late differentiation. 1,25-Dihydroxyvitamin D(3) functions by interacting with an intracellular receptor that binds to DNA at vitamin D response elements (VDRE) thereby affecting transcription. 1,25-Dihydroxyvitamin D(3) has been demonstrated to alter the expression of several enzymes involved in signal transduction, and presumably this is the mechanism through which the hormone regulates differentiation. It has recently been shown that 1,25-dihydroxyvitamin D(3) specifically increases the expression/activity of phospholipase D-1, an enzyme that hydrolyzes phospholipids to generate lipid messengers, such as diacylglycerol (DAG). DAG, in turn, is known to activate several members of the protein kinase C (PKC) family. It has been proposed that this signaling pathway mediates late differentiation events in epidermal keratinocytes. In this article the data supporting a role for PKC and phospholipase D in keratinocyte differentiation, as well as in the pathogenesis of skin diseases, are reviewed and a model is proposed for the signaling pathways that regulate this process upon exposure to 1,25-dihydroxyvitamin D(3).

ABNORMAL AQUAPORIN-3 PROTEIN EXPRESSION IN HYPERPROLIFERATIVE SKIN DISORDERS

Non-melanoma skin cancers (NMSCs) and psoriasis represent common hyperproliferative skin disorders, with approximately one million new NMSC diagnoses each year in the United States alone and a psoriasis prevalence of about 2% worldwide. We recently demonstrated that the glycerol channel, aquaporin-3 (AQP3) and the enzyme phospholipase D2 (PLD2) interact functionally in epidermal keratinocytes of the skin to inhibit their proliferation. However, others have suggested that AQP3 is pro-proliferative in keratinocytes and is upregulated in the NMSC, squamous cell carcinoma (SCC). To evaluate the AQP3/PLD2 signaling module in skin diseases, we determined their levels in SCC, basal cell carcinoma (BCC) and psoriasis as compared to normal epidermis. Skin biopsies with the appropriate diagnoses (10 normal, 5 SCC, 13 BCC and 10 plaque psoriasis samples) were obtained from the pathology archives and examined by immunohistochemistry using antibodies recognizing AQP3 and PLD2. In normal epidermis AQP3, an integral membrane protein, was localized mainly to the plasma membrane and PLD2 to the cell periphery, particularly in suprabasal layers. In BCC, AQP3 and PLD2 levels were reduced as compared to the normal-appearing overlying epidermis. In SCC, AQP3 staining was “patchy,” with areas of reduced AQP3 immunoreactivity exhibiting positivity for Ki67, a marker of proliferation. PLD2 staining was unchanged in SCC. In psoriasis, AQP3 staining was usually observed in the cytoplasm rather than in the membrane. Also, in the majority of psoriatic samples, PLD2 showed weak immunoreactivity or aberrant localization. These results suggest that abnormalities in the AQP3/PLD2 signaling module correlate with hyperproliferation in psoriasis and the NMSCs.