Paola D'Andrea

Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness

Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The specific role of connexin channels in the homeostasis of different organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non‐syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild‐type (HCx26wt) and the F83L polymorphism, found in unaffected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water‐injected controls. In contrast, most recessive Cx26 mutations (identified in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identified as a polymorphism in heterozygous unaffected individuals, was devoid of function and thus may be pathologically significant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage‐gating properties were indistinguishable from those of HCx26wt. The identification of functional differences of disease causing mutations may lead to define which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1.

Hearing loss: frequency and functional studies of the most common connexin26 alleles

Mutations in the GJB2 gene, encoding the gap-junction channel protein connexin 26, account for the majority of recessive forms and some of the dominant cases of deafness. Here, we report the frequency of GJB2 alleles in the Italian population affected by hearing loss and the functional analysis of six missense mutations. Genetic studies indicate that, apart from the common 35delG, only few additional mutations can be detected with a significant frequency in our population. Transfection of communication-incompetent HeLa cells with Cx26 missense mutations revealed three distinct classes of functional deficits in terms of protein expression, subcellular localisation and/or functional activity. Moreover, the M34T mutant acted as a dominant inhibitor of wild-type Cx26 channel activity when the two proteins were co-expressed in a manner mimicking a heterozygous genotype. These data support the hypothesis of a functional role for M34T as a dominant allele and represent a further step towards a complete understanding of the role of GJB2 in causing hearing loss.

Dual mechanism of intercellular communication in HOBIT osteoblastic cells: a role for gap-junctional hemichannels.

Intercellular communication allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Paracrine stimulation and cell‐to‐cell coupling through gap junctions induce the formation of complex cellular networks, which favors the intercellular exchange of nutrients and second messengers. Intercellular Ca2+ signaling was investigated in human osteoblast‐like initial transfectant (HOBIT) cells, a human osteoblastic cell line in which cells retain most of the osteoblastic differentiation markers. HOBIT cells express connexin43 (Cx43) clustered at the cell‐to‐cell boundary and display functional intercellular coupling as assessed by the intercellular transfer of Lucifer yellow. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading and dye transfer were inhibited by 18α‐glycyrrhetinic acid (18‐GA), showing the involvement of gap junctions in signal propagation. Pretreatment of cells with suramin or with apyrase decreased the extent of wave propagation, suggesting that ATP‐mediated paracrine stimulation contribute to cell‐to‐cell signaling. The functional expression of gap‐junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake, and intracellular Ca2+ release, activated by uptake of inositol 1,4,5‐trisphosphate (InsP3) from the external medium. Gap‐junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18‐GA. A role for Cx hemichannels in adenosine triphosphate (ATP) release and paracrine stimulation is suggested.

Culture and differentiation of chondrocytes entrapped in alginate gels

SummaryWe studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.

Mechanism of mechanically induced intercellular calcium waves in rabbit articular chondrocytes and in HIG-82 synovial cells.

Intercellular communication through gap junctions allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Intercellular Ca2+ signaling was investigated with digital fluorescence video imaging in primary cultures of articular chondrocytes and in HIG‐82 synovial cells. In both cell types, mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18α‐glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+, mechanical stimulation induced communicated Ca2+ waves similar to controls; however, the number of HIG‐82 cells recruited decreased significantly. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte and HIG‐82 cell, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Treatment of cells with thapsigargin and with the phospholipase C (PLC) inhibitor U73122 blocked mechanically induced signal propagation. These results provide evidence that in chondrocytes and in HIG‐82 synovial cells, mechanical stimulation activates PLC, thus leading to an increase of intracellular inositol 1,4,5‐trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighboring cells. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate tissue responses in joint physiology.

Propagation of intercellular Ca2+ waves in mechanically stimulated articular chondrocytes

Intercellular Ca2+ signalling in primary cultures of articular chondrocytes was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18α‐glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+ mechanical stimulation failed to induce Ca2+ responses and communicated Ca2+ waves. Under these conditions Ca2+ microinjection induced intercellular waves involving the cells immediately surrounding the stimulated one. Mechanical stress induced Ca2+ influx in the stimulated, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cell treatment with thapsigargin failed to block mechanically induced signal propagation, but significantly reduced the number of cells involved in the communicated Ca2+ wave. Similar results were obtained with the phospholipase C inhibitor U73122, which is known to prevent InsP3 generation. These results provide evidence that mechanical stimulation induces a cytosolic Ca2+ increase that may permeate gap junctions, thus acting as an intercellular messenger mediating cell‐to‐cell communication in articular chondrocytes.

Extracellular ATP stimulates the early growth response protein 1 (Egr-1) via a protein kinase C-dependent pathway in the human osteoblastic HOBIT cell line.

Extracellular nucleotides exert an important role in controlling cell physiology by activating intracellular signalling cascades. Osteoblast HOBIT cells express P2Y(1) and P2Y(2) G-protein-coupled receptors, and respond to extracellular ATP by increasing cytosolic calcium concentrations. Early growth response protein 1 (Egr-1) is a C(2)H(2)-zinc-finger-containing transcriptional regulator responsible for the activation of several genes involved in the control of cell proliferation and apoptosis, and is thought to have a central role in osteoblast biology. We show that ATP treatment of HOBIT cells increases Egr-1 protein levels and binding activity via a mechanism involving a Ca(2+)-independent protein kinase C isoform. Moreover, hypotonic stress and increased medium turbulence, by inducing ATP release, result in a similar effect on Egr-1. Increased levels of Egr-1 protein expression and activity are achieved at very early times after stimulation (5 min), possibly accounting for a rapid way for changing the osteoblast gene-expression profile. A target gene for Egr-1 that is fundamental in osteoblast physiology, COL1A2, is up-regulated by ATP stimulation of HOBIT cells in a timescale that is compatible with that of Egr-1 activation.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: Evidence for an autoregulatory loop

The Early Growth Response protein (Egr-1) is a C2H2–zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation–reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H2O2 stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H2O2 stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Interleukin-1β Increases the Functional Expression of Connexin 43 in Articular Chondrocytes: Evidence for a Ca2+-Dependent Mechanism

Cell‐to‐cell interactions and gap junctions‐dependent communication are crucially involved in chondrogenic differentiation, whereas in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 (Cx43) and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of Interleukin‐1 (IL‐1) in the synovial fluid. We have examined the effects of IL‐1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL‐1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose‐dependent increase in the expression of Cx43 and by an enhanced Cx43 immunostaining at sites of cell‐to‐cell contact. IL‐1 stimulation induced a dose‐dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL‐1‐dependent up‐regulation of Cx43 could be prevented by intracellular Ca2+ chelation but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of Cx43. IL‐1 is one of the most potent cytokines that promotes cartilage catabolism; its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.