Laird D. Madison

Prestin is the motor protein of cochlear outer hair cells.

The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a ‘gating current’, which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.

Expression of estrogen receptor β in prostate carcinoma cells inhibits invasion and proliferation and triggers apoptosis

The involvement of estrogen receptor beta (ERβ) in prostate carcinogenesis has been hypothesized. Several reports have shown that ERβ expression was decreased when prostate cells undergo neoplastic transformation, suggesting that it could play a tumor‐suppressor role. By restoring ERβ expression in prostatic carcinoma cells by adenoviral delivery, we aimed to test this hypothesis. We observed that ERβ strongly inhibited the invasiveness and the growth of these cells. In addition, ERβ cells were undergoing apoptosis, as shown by quantification of Bax, poly(ADP‐ribose) polymerase and caspase‐3 expression. Our data suggest that ERβ acts as a tumor‐suppressor by its anti‐proliferative, anti‐invasive and pro‐apoptotic properties.

Prestin topology: localization of protein epitopes in relation to the plasma membrane.

Computer modeling of the outer hair cell (OHC) motor protein prestin produces ambiguous results regarding transmembrane regions and localization of its termini. To determine the location of prestins N- and C-termini, we created prestin constructs with synthetic epitopes located immediately upstream or downstream of prestin. The spatial distribution of these epitopes was studied in prestin-transfected cells using immunofluorescence. In permeabilized cells, antibodies label the plasma membrane of 30% of the cells, reflecting transfec- tion efficiency. Under non-permeabilizing conditions, the few labeled cells also displayed a lack of plasma membrane integrity. These data suggest that prestins N-and C-termini are cytoplasmic. Furthermore, prestin staining in OHCs was observed only under permeabilizing conditions. These results implicate prestins N- and C-termini as portions that may interact with other cytoplasmic proteins. A model of prestin membrane topology is also considered based on the results.

Insulin-Like Growth Factor I Production Is Essential for Anabolic Effects of Thyroid Hormone in Osteoblasts†

Thyroid hormone (T3) and insulin‐like growth factor I (IGF‐I) are critical regulators of skeletal function. T3 increases IGF‐I production in bone. To assess the potential role of IGF‐I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3‐E1 cells, exposed to T3 alone or under conditions that interfere with IGF‐I actions. T3 significantly increased osteoblast 3H‐proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both αIR3, a neutralizing monoclonal antibody to the IGF‐I receptor, and JB1, an IGF‐I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS‐ODN) to the IGF‐I receptor gene. Both IGF‐I and T3 had mitogenic effects that were inhibited by the antagonists. IGF‐I by itself did not stimulate 3H‐proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF‐I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses. (J Bone Miner Res 2000;15:188–197)

Prestin, the Motor Protein of Outer Hair Cells

Prestin is a gene recently cloned from mammalian cochlear outer hair cells (OHC) using a single cell type, outer minus inner hair cell, specific suppressive subtractive hybridization procedure. The localization and gene expression profile of the prestin protein fits the pattern of OHC’s development of electromotility. When prestin is abundantly expressed in normally nonmotile kidney cells, nonlinear capacitance and motility that are normally only seen in OHCs can be recorded. Furthermore, both nonlinear capacitance and motility can be reduced by salicylate, a well-known inhibitor of electromotility. These data suggest that prestin is the motor protein of OHCs. Amino acid sequence and gene structure analysis indicate that prestin is the fifth member of a newly discovered anion transport family (SLC26) that includes PDS, DRA and DTDST, which are chloride-iodide transporters, Cl–/HCO–3 exchangers or sulfate transporters. Prestin shares overall structure similarity with this anion transporter family. Recently, intracellular anions (chloride or bicarbonate) were found to be essential for OHC electromotility and prestin’s function.

The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting

Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cells membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G/Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestins function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function.

N-linked glycosylation sites of the motor protein prestin: effects on membrane targeting and electrophysiological function.

Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N‐glycoprotein with three potential N‐linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N‐glycosylation were investigated by producing single‐point (N163Q and N166Q) or double‐point mutations (NN163/166QQ and NN163/166AA) at putative N‐glycosylation sites. Further, treatment with tunicamycin or glycopeptidase‐F was used to determine the consequences of removing N‐linked glycosylation in wild‐type prestin. We determined the effects of these manipulations on prestins cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell‐types. Data indicate that prestin is a glycoprotein with N‐linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N‐linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non‐linear capacitance, a signature of prestins motor function. Compared to glycosylated prestin, the fully de‐glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.

Genomic characterization and expression of mouse prestin, the motor protein of outer hair cells.

We previously identified the gerbil gene (gPres) that encodes prestin, the putative motor protein responsible for outer hair cell (OHC) electromotility. Here we report the cloning and characterization of the complete genomic structure of the mouse Prestin (mPres) gene. We performed 5′- and 3′-RACE to determine the size and identity of the full-length mRNA transcript. The mPres gene encodes a protein 96% identical to the gPres gene product. The prestin open reading frames are 91% identical at the nucleotide level. Using an antibody raised against the N-terminus of gerbil Prestin, we observed mPrestin expression by immunofluorescence in the lateral membrane of mouse OHCs and found no detectable expression elsewhere in the organ of Corti. On the basis of the available genomic sequence from mouse Chromosome (Chr) 5, we concluded that the mPres gene is centromerically related to and resides within 19 kb of, the Reln gene. We were also able to characterize the exon/intron junctions of mPres by using cDNA/genomic sequence comparisons, as well as exon-exon PCR and sequencing. The mPres gene has 18 exons that encode protein and two exons in the 5′ UTR. A CpG island, located at the start of exon 1, is a potential transcription start site. Sequence analysis of the ~500 bp upstream from exon 1 revealed multiple potential transcription factor binding sites, including both TATA and GC boxes, as well as other regulatory-element binding sites.

An osteoporosis and fracture intervention program increases the diagnosis and treatment for osteoporosis for patients with minimal trauma fractures.

BACKGROUND As fewer than 25% of patients with an osteoporotic minimal trauma fracture (MTF) are evaluated and treated for osteoporosis, an osteoporosis and fracture intervention program (OFIP) was developed. METHODS Patients hospitalized with MTF were educated about and treated for osteoporosis and were evaluated by the osteoporosis team at 6 and 12 months after discharge. Patients seen in the emergency department were given information about osteoporosis and encouraged to seek medical care at the osteoporosis office. RESULTS While 165 patients hospitalized with an MTF participated in the OFIP, 38 patients received routine osteoporosis education. At the 6-month follow-up, in the OFIP group, 68% of patients with hip fracture and 54% of patients with non-hip fracture were taking antiresorptive medications. There was no change in treatment rate among patients receiving conventional care. CONCLUSIONS The rates of diagnosis of osteoporosis and treatment implementation following an MTF increased when the intervention occurred at the time of hospitalization.

Identification of Differentially Expressed cDNA Clones from Gerbil Cochlear Outer Hair Cells

In order to identify genes that are associated with outer hair cell(OHC)-specific function, a plasmid library enriched with OHC-specific gene products was constructed using single cell-type-specific complementary DNA (cDNA) and a PCR subtractive hybridization strategy. As a first step, we created separate OHC and inner hair cell (IHC) cDNA pools from individually collected cells using a nonspecific reverse transcription polymerase chain reaction. Next, the OHC cDNA was subtracted against IHC cDNA using a PCR-based subtractive technique. IHCs and OHCs share many common features, making IHC cDNA an ideal ‘driver’ to ‘subtract away’ common hair cell gene products and enrich differentially expressed cDNAs, including OHC-specific genes. The subtracted OHC cDNAs were then cloned to generate an OHC – IHC subtracted cDNA plasmid library. Finally, a differential screening procedure was performed, resulting in 477 differentially positive clones. After analysis of these 477 clones, 50 known genes were identified, including two previously known OHC-specific proteins: oncomodulin and the recently described motor protein prestin. An additional 84 novel clones were also found. As this library of cDNA fragments represents differentially expressed genes in OHCs, it can be used as starting material for isolation and characterization of a complete set of OHC gene products, an important step in investigating normal and abnormal cochlear function.