Jonathan H. Zippin

Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains

Intracellular targets of the ubiquitous second messenger cAMP are located at great distances from the most widely studied source of cAMP, the G protein responsive transmembrane adenylyl cyclases. We previously identified an alternative source of cAMP in mammalian cells lacking transmembrane spanning domains, the “soluble” adenylyl cyclase (sAC). We now demonstrate that sAC is distributed in specific subcellular compartments: mitochondria, centrioles, mitotic spindles, mid‐bodies, and nuclei, all of which contain cAMP targets. Distribution at these intracellular sites proves that adenylyl cyclases are in close proximity to all cAMP effectors, suggesting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases targeted to specific microdomains throughout the cell.

Bicarbonate-responsive "soluble" adenylyl cyclase defines a nuclear cAMP microdomain.

Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

CO2/HCO3--responsive soluble adenylyl cyclase as a putative metabolic sensor

Cyclic AMP (cAMP) is an evolutionarily conserved regulator of metabolism. Recently, we identified a novel mammalian source of cAMP - soluble adenylyl cyclase (sAC) - that is regulated directly by bicarbonate ions (HCO(3)(-)). As the concentration of HCO(3)(-) reflects cellular levels of carbon dioxide (CO(2)), energy-generating metabolic processes (which increase intracellular CO(2)) are poised to activate bicarbonate-responsive sAC. This direct link between metabolic activity, sAC and cAMP could represent an evolutionarily conserved mechanism of metabolic feedback regulation.

Soluble adenylyl cyclase mediates nerve growth factor-induced activation of Rap1.

Nerve growth factor (NGF) and the ubiquitous second messenger cyclic AMP (cAMP) are both implicated in neuronal differentiation. Multiple studies indicate that NGF signals to at least a subset of its targets via cAMP, but the link between NGF and cAMP has remained elusive. Here, we have described the use of small molecule inhibitors to differentiate between the two known sources of cAMP in mammalian cells, bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC) and G protein-regulated transmembrane adenylyl cyclases. These inhibitors, along with sAC-specific small interfering RNA, reveal that sAC is uniquely responsible for the NGF-elicited rise in cAMP and is essential for the NGF-induced activation of the small G protein Rap1 in PC12 cells. In contrast and as expected, transmembrane adenylyl cyclase-generated cAMP is responsible for Rap1 activation by the G protein-coupled receptor ligand PACAP (pituitary adenylyl cyclase-activating peptide). These results identify sAC as a mediator of NGF signaling and reveal the existence of distinct pathways leading to cAMP-dependent signal transduction.

CO2/HCO3−- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

Background: The affinity of soluble adenylyl cyclase (sAC) for its substrate ATP suggested that it might be sensitive to fluctuations in ATP. Results: In sAC-overexpressing and glucose-responsive cells, sAC-generated cAMP reflects intracellular ATP levels. Conclusion: sAC can be an ATP sensor inside cells. Significance: sAC serves as a metabolic sensor via regulation by three cellular metabolites: ATP, bicarbonate, and calcium. The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

Soluble Adenylyl Cyclase Defines a Nuclear cAMP Microdomain in Keratinocyte Hyperproliferative Skin Diseases

Cyclic adenosine monophosphate (cAMP) is a nearly ubiquitous signaling molecule important for numerous signaling pathways in human skin. We studied a novel class of mammalian adenylyl cyclase, the soluble adenylyl cyclase (sAC). We examined sAC localization in normal human skin and found it to be present in keratinocytes, melanocytes, mononuclear cells, eccrine ducts, and nerves. In normal skin, sAC keratinocyte staining was evenly distributed throughout the cell. However, in certain hyperproliferative disorders of the skin, including psoriasis, verruca vulgaris, and SCCIS on sun-damaged skin, sAC keratinocyte staining was predominantly nuclear. In contrast, in other hyperproliferative disorders, such as basal cell carcinoma, sAC staining was similar to normal human skin. Using a model of epithelial differentiation, we established that sAC migrates into the nucleus when differentiated cells are induced to reenter the cell cycle. Previous work had determined that nuclear sAC activates the cAMP-response-element-binding (CREB) transcription factor, and we found that in psoriasis lesions, nuclear sAC occurs concomitantly with activation of CREB. Hence, sAC may play a role in the pathogenesis of certain hyperproliferative skin disorders via modulation of gene expression.

Type 10 soluble adenylyl cyclase is overexpressed in prostate carcinoma and controls proliferation of prostate cancer cells

Background: Soluble adenylyl cyclase (sAC) may be an alternative intracellular localized source of cAMP controlling proliferation. Results: sAC is overexpressed in prostate carcinoma, and inhibition of sAC leads to cell cycle arrest. Conclusion: sAC controls proliferation of prostate carcinoma cells. Significance: sAC represents a novel pathway promoting proliferation in cancer cells and is a promising target for prostate cancer treatment. cAMP signaling plays an essential role in modulating the proliferation of different cell types, including cancer cells. Until now, the regulation of this pathway was restricted to the transmembrane class of adenylyl cyclases. In this study, significant overexpression of soluble adenylyl cyclase (sAC), an alternative source of cAMP, was found in human prostate carcinoma, and therefore, the contribution of this cyclase was investigated in the prostate carcinoma cell lines LNCaP and PC3. Suppression of sAC activity by treatment with the sAC-specific inhibitor KH7 or by sAC-specific knockdown mediated by siRNA or shRNA transfection prevented the proliferation of prostate carcinoma cells, led to lactate dehydrogenase release, and induced apoptosis. Cell cycle analysis revealed a significant rise in the G2 phase population 12 h after sAC inhibition, which was accompanied by the down-regulation of cyclin B1 and CDK1. sAC-dependent regulation of proliferation involves the EPAC/Rap1/B-Raf signaling pathway. In contrast, protein kinase A does not play a role. In conclusion, this study suggests a novel sAC-dependent signaling pathway that controls the proliferation of prostate carcinoma cells.

Soluble Adenylyl Cyclase Antibody Profile as a Diagnostic Adjunct in the Assessment of Pigmented Lesions

OBJECTIVE To investigate the usefulness of a novel marker for melanocytic proliferations. DESIGN Using a novel monoclonal antibody against soluble adenylyl cyclase (sAC), various benign and malignant melanocytic proliferations were immunostained. SETTING Weill Medical College of Cornell University dermatopathology laboratory. MAIN OUTCOME MEASURES The results were qualitative, not quantifiable. RESULTS The sAC immunostaining produced distinctive patterns that paralleled melanomagenesis. At one pole of the spectrum were benign nevi, including atypical nevi of special sites and recurrent nevi showing a distinct pattern of dotlike Golgi staining, while at the opposite pole was melanoma, in which many cells demonstrated an intense pannuclear expression pattern, often accompanied by loss of the Golgi expression pattern. Melanomas of lentigo maligna and acral lentiginous subtypes exhibited the most striking pannuclear expression, while nodular melanomas showed the least, although with supervening enhanced diffuse cytoplasmic expression. Loss of the Golgi expression pattern was a feature of malignant melanoma. CONCLUSION The sAC expression pattern is complex but seems discriminatory, with distinctive and variable staining patterns according to the nature of the lesion biopsied.

Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally within independently-regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP; G protein regulated transmembrane adenylyl cyclases and bicarbonate- calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, understanding cAMP signaling demands methods to distinguish between them. We developed a mass spectrometry based adenylyl cyclase assay which we used to identify a novel sAC-specific inhibitor, LRE1. LRE1 binds to the bicarbonate activator binding site and inhibits sAC via a unique allosteric mechanism. LRE1 prevents sAC-dependent processes in cellular and physiological systems and facilitates exploration of the therapeutic potential of sAC inhibition.

Allergic contact dermatitis to cosmetics.

Allergic contact dermatitis caused by cosmetic products is an increasing concern given the continual creation and introduction of new cosmetics to the public. This article presents an overview of how to evaluate a patient for patch testing, including common areas for cosmetic-induced dermatitis, common cosmetic allergens, and proper management.