Malcolm B. Hodgins

Human diseases: clues to cracking the connexin code?

The vertebrate gap junctions formed by the connexin family of transmembrane proteins came to the attention of geneticists in 1993 with the identification of mutations linked to a form of demyelinating neuropathy. Since then, several other genetic disorders have been linked to mutations in specific connexin genes. Also, different diseases can result from different mutations in the same connexin gene. In addition, specific connexin knockout mice have surprising phenotypes. This is leading cell biologists to look afresh at connexins and their involvement in intercellular communication through gap junctions, a process that seems central to coordinating cell function within tissues. Here, we comment on how genetic studies are giving a new impetus to the cell biology of gap junctions.

An immunohistochemical study of androgen, oestrogen and progesterone receptors in the vulva and vagina.

Objective Tomap potential sites of sex steroid action in the human vulva.

Changing patterns of gap junctional intercellular communication and connexin distribution in mouse epidermis and hair follicles during embryonic development

In the mouse embryo between embryonic days 12 (E12) and 16, regular arrays of epidermal placodes on the mystacial pad develop into whisker follicles. This system was chosen for analysis of gap junctional intercellular communication during differentiation. The patterns of communication were studied by microinjection of the tracers Lucifer yellow‐CH (LY‐CH) and neurobiotin (NB), while immunofluorescent staining was used to study distribution of connexins 26 and 43. Extensive communication was seen between keratinocytes in developing hair pegs or, in later‐stage hair follicles, in the germinative matrix. Coupling between adjacent hair pegs via interfollicular epidermis was not observed. Coupling also became restricted as follicular cells differentiated to form outer root sheath, inner root sheath, and hair shaft. Extensive gap junctional coupling is characteristic of keratinocytes that are rapidly proliferating (as in hair pegs and germinative matrix). Follicular keratinocytes commence differentiation shortly before restriction of gap junctional coupling becomes evident. Dermal mesenchymal cells undergoing different modes of differentiation also exhibit differences in gap junctional coupling, as evidenced by poor transfer of LY‐CH between cells in dermal condensations of hair follicles compared with extensive transfer elsewhere in the dermis. LY‐CH and NB were not transferred between epidermal or follicular epithelium and mesenchyme, arguing against a direct role for gap junctions permeable to known second messenger molecules or nucleotides in epithelial‐mesenchymal interactions in this system. The distribution of connexins 26 and 43 in epidermis and hair follicles changed during differentiation but there was no correlation with changing patterns of dye transfer, indicating an unexpected degree of complexity in the relationship between gap junctional intercellular communication and connexin protein distribution during development. Dev. Dyn. 1997;210:417–430.

The relationship between connexins, gap junctions, tissue architecture and tumour invasion, as studied in a novel in vitro model of HPV-16-associated cervical cancer progression.

Disruption of gap junctional intercellular communication (GJIC) and/or connexins (gap junction proteins) is frequently reported in malignant cell lines and tumours. Certain human papillomaviruses (HPV) associated with the development of cancers, especially of the cervix, have previously been reported to downregulate GJIC in vitro. There is also evidence for reduced gap junctions in cervical dysplasia. However, many squamous hyperproliferative conditions, including HPV-induced warts, often show extensive upregulation of certain connexins. The association between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in cervical tumour progression, remains unclear. Therefore, using a variety of nonmalignant and malignant cell lines and an organotypic raft-culture system, we investigated the relationship between HPV, gap junctions and tumour progression. Established cervical tumour cell lines carrying HPV were unable to communicate via gap junctions (when assayed by dye-transfer techniques). This correlated with lack of connexin protein expression, while transfection with connexins 26 or 43 led to functional gap junction membrane plaques. On the other hand, immortal but nonmalignant cell lines that contained episomal or integrated HPV-16, but required feeder-layer and growth-factor support, were consistently well coupled, and expressed multiple connexins at membrane junctions. In vitro selection of feeder-layer and growth-factor-independent variants eventually lead to loss of GJIC, which correlated with loss of membrane and increased cytoplasmic connexin 43 localization. However, this was preceded by loss of differentiation and stromal invasion, as assayed on the organotypic raft-culture model. Using this model, a comparison between noncoupled, well-coupled and connexin-transfected cell lines revealed no firm correlation between GJIC and dysplasia, but GJIC appeared to favour increased stratification. These findings demonstrate that loss of GJIC is frequent, but appears to occur more as a consequence of, rather than being the cause of, epithelial dysplasia, and may be influenced by, but is not directly attributable to, HPV.

Transport and Function of Cx26 Mutants Involved in Skin and Deafness Disorders

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and ΔE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and ΔE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor ΔE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and ΔE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.

Polyamines regulate gap junction communication in connexin 43-expressing cells

The control of cell-cell communication through gap junctions is thought to be crucial in normal tissue function and during various stages of tumorigenesis. However, few natural regulators of gap junctions have been found. We show here that increasing the activity of ornithine decarboxylase, or adding polyamines to the outside of cells, increases the level of gap junction communication between various epithelial cells. Conversely, reduction of ornithine decarboxylase activity decreases the level of gap junction communication. This regulation is dependent upon the expression of connexin 43 (Cx43 or Cxalpha1), which is a major connexin expressed in many different cell types, and involves an increase in Cx43 and its cellular re-distribution.

Nature of Cx30-containing channels in the adult mouse mammary gland

Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse.

The Effects of a Mutant Connexin 26 on Epidermal Differentiation

To elucidate the mode of action of dominant mutant connexins in causing inherited skin diseases, transgenic mice were produced that express the true Vohwinkel syndrome-associated mutant Cx26 (D66H), from a keratin 10 promoter, specifically in the suprabasal epidermal keratinocytes. Following birth, the transgenic mice developed keratoderma similar to that of human carriers of Cx26 (D66H). Expression of the transgene resulted in a loss of Cx26 and Cx30 at intercellular junctions of epidermal keratinocytes and accumulation of these connexins in the cytoplasm. Injection of primary mouse keratinocytes with Lucifer Yellow showed no difference in terms of dye spreading between transgenic and non transgenic keratinocytes in vitro. Expression of the mutant Cx26 (D66H) did not interfere with the formation of the epidermal water barrier during late embryonic development. Attempts to produce transgenic mice expressing the wild type form of Cx26 from the K10 promoter failed to produce viable animals although transgenic embryos were recovered at days 9 and 12 of gestation, suggesting that the transgene might be embryonic lethal.

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.