HCN1 and HCN2 proteins are expressed in cochlear hair cells: HCN1 can form a ternary complex with protocadherin 15 CD3 and F-actin-binding filamin A or can interact with HCN2.

A unique coupling between HCN1 and stereociliary tip-link protein protocadherin 15 has been described for a teleost vestibular hair-cell model and mammalian organ of Corti (OC) (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227-3238). We now show that Ca(2+)-dependent interaction of the organ of Corti HCN1 and protocadherin 15 CD3 is mediated by amino-terminal sequence specific to HCN1 and is not replicated by analogous specific peptides for HCN2 or HCN4 nor by amino-terminal sequence conserved across HCN isoforms utilized in channel formation. Furthermore, the HCN1-specific peptide binds both phosphatidylinositol (3,4,5)-trisphosphate and phosphatidylinositol (4,5)-bisphosphate but not phosphatidylinositol 4-phosphate. Singly isolated cochlear inner and outer hair cells express HCN1 transcript, and HCN1 and HCN2 protein is immunolocalized to hair-cell stereocilia by both z-stack confocal and pre-embedding EM immunogold microscopy, with stereociliary tip-link and subcuticular plate sites. Quantitative PCR indicates HCN1/HCN2/HCN3/HCN4 = 9:9:1:89 in OC of the wild-type mouse, with HCN4 protein primarily attributable to inner sulcus cells. A mutant form of HCN1 mRNA and protein is expressed in the OC of an HCN1 mutant, corresponding to a full-length sequence with the in-frame deletion of pore-S6 domains, predicted by construct. The mutant transcript of HCN1 is ∼9-fold elevated relative to wild-type levels, possibly representing molecular compensation, with unsubstantial changes in HCN2, HCN3, and HCN4. Immunoprecipitation protocols indicate alternate interactions of full-length proteins; HCN1 can interact with protocadherin 15 CD3 and F-actin-binding filamin A forming a complex that does not include HCN2, or HCN1 can interact with HCN2 forming a complex without protocadherin 15 CD3 but including F-actin-binding fascin-2.

CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1.

The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic C-terminus of CNGA3 was found by yeast two-hybrid analysis to bind the C-terminus of EMILIN1 (elastin microfibril interface-located protein 1) in both the vestibular hair cell model and rat organ of Corti. Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca2+ with Kd=1.6×10-6 M for trout hair cell proteins and Kd=2.7×10-7 M for organ of Corti proteins at 68 µM Ca2+. Pull-down assays indicated that the binding to organ of Corti CNGA3 was attributable to the EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the C-terminus. Saccular hair cells also express the transcript for PDE6C (phosphodiesterase 6C), which in cone photoreceptors regulates the degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and ß1 integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.

Cell cycle and apoptosis regulatory protein (CARP)-1 is a novel, adriamycin-inducible, diffuse large B-cell lymphoma (DLBL) growth suppressor.

UNLABELLED: Diffuse large B-cell lymphoma (DLCL) accounts for 30-40% of adult non-Hodgkin's Lymphoma (NHL). Current anti-NHL therapies often target cellular growth suppression pathways and include R-CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone plus monoclonal anti-CD20 antibody rituximab). However, since many patients relapse, resistant cells to these therapies remain a significant problem and necessitate development of new intervention strategies. Cell cycle and apoptosis regulatory protein (CARP)-1 functions in a biphasic manner to regulate growth factor as well as chemotherapy (adriamycin, etoposide, or iressa)-dependent signaling. PURPOSE: To determine whether CARP-1 is a novel suppressor of lymphoma growth. METHODS: Flow cytometric analyses coupled with Western immunoblotting, cell growth, apoptosis, and immunocytochemistry methodologies were utilized to determine CARP-1-dependent lymphoma growth inhibition in vitro and in vivo. RESULTS: CARP-1 expression correlated with activated caspase-3 and inversely correlated with activated Akt in DLCL. Exposure to adriamycin stimulated CARP-1 expression and inhibited growth of Raji cells, but not CHOP-resistant WSU-DLCL2 cells. Expression of wild-type CARP-1 or its apoptosis-inducing mutants inhibited growth of Raji as well as CHOP-resistant WSU-DLCL2 cells, in part by activating caspase-9 and apoptosis. Since CARP-1 harbors multiple, apoptosis-promoting subdomains, we investigated whether epigenetic compensation of CARP-1 function by intracellular delivery of trans-activator of transcription (TAT) domain-tagged CARP-1 peptide(s) will inhibit lymphoma growth. Treatments with TAT-tagged CARP-1 peptides suppressed growth of the Raji and WSU-DLCL2 cells by stimulating apoptosis. TAT-CARP-1 (1-198) as well as (896-1150) peptides also suppressed growth of WSU-DLCL2 cell-derived tumor xenografts in SCID mice, while administration of TAT-CARP-1 (1-198) also inhibited growth of WSU-FSCCL cell-derived ascites and prolonged host survival. CONCLUSION: CARP-1 is a suppressor of NHL growth and could be exploited for targeting the resistant DLCL.

Calcium-dependent binding of HCN1 channel protein to hair cell stereociliary tip link protein protocadherin 15 CD3.

The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca(2+). In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K(D) = 2.39 x 10(-7) m. Ca(2+) at 26.5-68.0 microm promoted binding, with K(D) = 5.26 x 10(-8) m (at 61 microm Ca(2+)). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession number XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca(2+)-dependent, although the binding was skewed toward a lower effective maximum [Ca(2+)] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 microm Ca(2+). Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells.

Immunohistochemical localization of adrenergic receptors in the rat organ of corti and spiral ganglion.

Alpha(1)-, beta(1)-, and beta(2)-adrenergic receptors (ARs), which mediate responses to adrenergic input, have been immunohistochemically identified within the organ of Corti and spiral ganglion with polyclonal antibodies of established specificity. Alpha(1)-AR was immunolocalized to sites overlapping supranuclear regions of inner hair cells as well as to nerve fibers approaching the base of inner hair cells, most evident in the basal cochlear turn. A similar preponderance across cochlear turns for alpha(1)-AR in afferent cell bodies in the spiral ganglion pointed to type I afferent dendrites as a possible neural source of alpha(1)-AR beneath the inner hair cell. Foci of immunoreactivity for alpha(1)-AR, putatively neural, were found overlapping supranuclear and basal sites of outer hair cells for all turns. Beta(1)- and beta(2)-ARs were immunolocalized to sites overlapping apical and basal poles of the inner and outer hair cells, putatively neural in part, with immunoreactive nerve fibers observed passing through the habenula perforata. Beta(1)- and beta(2)-ARs were also detected in the cell bodies of Deiters' and Hensen's cells. Within the spiral ganglion, beta(1)- and beta(2)-ARs were immunolocalized to afferent cell bodies, with highest expression in the basal cochlear turn, constituting one possible neural source of receptors within the organ of Corti, specifically on type I afferent dendrites. Beta(1)- and beta(2)-ARs in Hensen's and Deiters' cells would couple to Galphas, known to be present specifically in the supporting cells. Overall, adrenergic modulation of neural/supporting cell function within the organ of Corti represents a newly considered mechanism for modifying afferent signaling.

Transactivator of transcription-tagged cell cycle and apoptosis regulatory protein-1 peptides suppress the growth of human breast cancer cells in vitro and in vivo.

Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)-tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1-198, 197-454, and 896-1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1-198(Y192/F)) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1-198(Y192/F)), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1-198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1-198(Y192/F)), inhibits growth of human breast cancer cell-derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing.

Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

Narrow-band UVB induces apoptosis in human keratinocytes.

Narrow-band ultraviolet (NB-UVB) phototherapy emits mostly 311/312 nm light and is commonly used in the treatment of inflammatory skin disorders. As a source of UVB irradiation, NB-UVB causes apoptosis in T lymphocytes but its effects on keratinocytes are unknown. Herein, we have investigated the ability of NB-UVB to induce apoptosis in keratinocytes. Two types of human keratinocytes, primary and immortalized, were exposed to NB-UVB and broad-band UVB (BB-UVB; 315-280 nm) and tested for apoptosis. Both UVB light sources induced apoptosis in keratinocytes as determined by the presence of DNA ladders, although NB-UVB required approximately ten fold higher doses; NB-UVB (1000 mJ/cm2) and BB-UVB (125 mJ/cm2). By comparison, lower doses of NB-UVB (750 mJ/cm2) induced apoptosis in T lymphocytes, suggesting cell type specificity for NB-UVB induced apoptosis. Approximately, 50% or more of the cells underwent apoptosis when exposed to NB-UVB or BB-UVB as revealed by TUNEL assay. Electron micrographs showed that NB-UVB irradiated keratinocytes contained marked chromatin condensation, extensive cytoplasmic vacuolization and fragmentation of the nuclear envelope. Furthermore, Western blot analysis confirmed the presence of activated products of caspase 3 in keratinocytes that received apoptotic doses of NB-UVB. This study defines conditions by which NB-UVB irradiation causes apoptosis in keratinocytes.

Folic acid reduces nuclear translocation of beta-catenin in rectal mucosal crypts of patients with colorectal adenomas.

We have demonstrated that folic acid inhibits cell proliferation and epidermal growth factor receptor (EGFR) activation in colon cancer cell lines. We examined the effect of one year supplemental folic acid (5 mg/day) on the rectal mucosal expression of beta-catenin and pGSK3beta, known to be affected by EGF-R, in patients with colorectal adenomas. Folic acid treatment significantly reduced nuclear expression of beta-catenin (P < 0.05) and cellular expression of pGSK3beta (P < 0.01) when compared to placebo. Folic acid may exert its chemopreventive effect, at least in part, through inhibition of nuclear translocation of beta-catenin.

Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437.

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.

Expression of EGF-receptor related protein (ERRP) decreases in gastric mucosa during aging and carcinogenesis.

Aging and gastrointestinal malignancies, including that of the stomach are associated with increased activation of EGF-receptor (EGFR). Although the intracellular events that regulate this process are poorly understood, we hypothesize that loss of ERRP (EGFR-related protein; GenBank accession number AF187818), a recently identified negative regulator of EGFR, that possesses a substantial homology to the ligand binding extracellular domain of EGFR, may contribute to this event. In support of our hypothesis, we have observed that in Fischer-344 rats, whereas aging is associated with increased activation of EGFR in the gastric mucosa, expression of ERRP decreases inthis tissue during this period. The latter is accompanied by a concomitant reduction in the amount of TGF-alpha bound to ERRP. In contrast, the amount of TGF-alpha bound to EGFR is found to be higher in the gastric mucosa of aged than in young rats. This is accompanied by a concomitant rise in EGFR levels. In the gastric mucosa, EGFR and ERRP are found to be colocalized. Gastric adenocarcinoma in humans, which has been shown to be associated with increased activation of EGFR, shows a substantial reduction in ERRP expression, when compared with benign tissues. We conclude that increased activation of EGFR in the gastric mucosa during aging and carcinogenesis may partly be due to the loss of ERRP.

Expression of subunits for the cAMP-sensitive 'olfactory' cyclic nucleotide-gated ion channel in the cochlea: implications for signal transduction.

Cyclic nucleotide-gated (CNG) ion channels have been implicated as functioning in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. The olfactory, cAMP-sensitive CNG ion channel in vivo is considered to comprise the pore-forming CNG2 subunit together with CNG5 and CNG4.3 modulatory subunits. The expression of these 'olfactory' CNG subunit transcripts in microdissected subfractions of the rat cochlea and hair cell libraries has been investigated with RT-PCR. Unmodified transcripts of CNG2 were detected in the organ of Corti, lateral wall and spiral ganglion subfractions. CNG5 message was found in both the sensory organ of Corti and the non-sensory lateral wall subfractions but not in the spiral ganglion subfraction. The CNG5 sequence obtained for the organ of Corti fraction encompassed 78% of the olfactory CNG5 cDNA sequence. CNG5 message has also been detected in an inner hair cell cDNA library. In the lateral wall, unmodified CNG5 sequence was observed as well as truncated versions of CNG5 transcripts, one of which was also found in the rat brain. The truncated versions were characterized by deletions that resulted in a shift in reading frame and the premature appearance of a stop codon. The 'olfactory' CNG4.3 cDNA was amplified from all three subfractions. Within the cochlea, CNG2 immunoreactivity was selectively distributed in a pattern similar to that of adenylyl cyclase type I. Immunoreactivity to CNG2 has been localized to stereocilia of inner hair cells. CNG5 immunoreactivity was associated with stereocilia and lateral plasma membranes of outer hair cells. We conclude that transcripts necessary for a functional cAMP-sensitive CNG ion channel are present in the cochlea resulting from combinations of CNG2 with CNG5 and CNG4.3. Further, the localization of CNG2 and CNG5 immunoreactivity to hair cell stereocilia suggests a role for cAMP-sensitive CNG channels in hair cell signal transduction.

Voltage-gated Ca2+ channel Ca(V)1.3 subunit expressed in the hair cell epithelium of the sacculus of the trout Oncorhynchus mykiss: cloning and comparison across vertebrate classes.

Full-length sequence (>6.5 kb) has been determined for the Ca(V)1.3 pore-forming subunit of the voltage-gated Ca(2+) channel from the saccular hair cells of the rainbow trout (Oncorhynchus mykiss). Primary structure was obtained from overlapping PCR and cloned fragments, amplified by primers based on teleost, avian, and mammalian sources. Trout saccular Ca(V)1.3 was localized to hair cells, as evidenced by its isolation from an epithelial layer in which the hair cell is the only intact cell type. The predicted amino acid sequence of the trout hair cell Ca(V)1.3 is approximately 70% identical to the sequences of avian and mammalian Ca(V)1.3 subunits and shows L-type characteristics. The trout hair cell Ca(V)1.3 expresses a 26-aa insert in the I-II cytoplasmic loop (exon 9a) and a 10-aa insert in the IVS2-IVS3 cytoplasmic loop (exon 30a), neither of which is appreciably represented in trout brain. The exon 9a insert also occurs in hair cell organs of chick and rat, and appears as an exon in human genomic Ca(V)1.3 sequence (but not in the Ca(V)1.3 coding sequence expressed in human brain or pancreas). The exon 30a insert, although expressed in hair cells of chick as well as trout, does not appear in comparable rat or human tissues. Further, the IIIS2 region shows a splice choice (exon 22a) that is associated with the hair cell organs of trout, chick, and rat, but is not found in human genomic sequence. The elucidation of the primary structure of the voltage-gated Ca(2+) channel Ca(V)1.3 subunit from hair cells of the teleost, representing the lowest of the vertebrate classes, suggests a generality of sensory mechanism for Ca(V)1.3 across hair cell systems. In particular, the exon 9a insert of this channel appears to be the molecular feature most consistently associated with hair cells from fish to mammal, consonant with the hypothesis that the latter region may be a signature for the hair cell.