Gap junction proteins are key drivers of endocrine function

    Gap junction proteins are key drivers of endocrine function Paolo Meda PII: DOI: Reference:

S0005-2736(17)30079-2 doi:10.1016/j.bbamem.2017.03.005 BBAMEM 82444

To appear in:

BBA - Biomembranes

Received date: Revised date: Accepted date:

16 December 2016 3 March 2017 6 March 2017

Please cite this article as: Paolo Meda, Gap junction proteins are key drivers of endocrine function, BBA - Biomembranes (2017), doi:10.1016/j.bbamem.2017.03.005

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ACCEPTED MANUSCRIPT GAP JUNCTION PROTEINS ARE KEY DRIVERS OF ENDOCRINE FUNCTION

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Paolo Meda

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Department of Cell Physiology and Metabolism, University of Geneva Medical School,

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Switzerland

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Keywords: hormones; insulin; renin; Cx36, Cx40, diabetes, hypertension

Corresponding

author:

Paolo

Meda,

M.D.

Phone:

+41793532215;

E-mail:

[email protected]

Paolo Meda

BBA Biomembranes, 2017

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ACCEPTED MANUSCRIPT Abstract It has long been known that the main secretory cells of exocrine and endocrine glands are connected by gap junctions, made by a variety of connexin species that ensure their electrical and metabolic coupling. Experiments in culture systems and animal models have since provided

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increasing evidence that connexin signaling contributes to control the biosynthesis and release

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of secretory products, as well as the life and death of secretory cells. More recently, genetic studies have further provided the first lines of evidence that connexins also control the function

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of human glands, which are central to the pathogenesis of major endocrine diseases. Here, we summarize the recent information gathered on connexin signaling in the latter systems, since the last reviews on the topic, with particular regard to the pancreatic beta cells which produce insulin, and the renal cells which produce renin. These cells are keys to the development of

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various forms of diabetes and hypertension, respectively, and combine to account for the

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exploding, worldwide prevalence of the metabolic syndrome.

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ACCEPTED MANUSCRIPT Contents Introduction 1. Connexin signaling in cells producing peptide hormones

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1.1. the β-cells of pancreas producing insulin

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1.2. the α-, δ-, PP- and ε-cells of pancreas producing glucagon, somatostatin, pancreatic

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polypeptide and ghrelin

1.3. the neurons of hypothalamus secreting GnRH, TRH, CRH, somatostatin, GHRH, dopamine, vasopressin, and oxytocin

1.4. the acidophil and basophil cells of the anterior pituitary producing GH, PRL, FSH, LH,

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TSH and ACTH

1.5. the B pinealocytes of pineal gland producing melatonin

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1.6. the melanotroph cells of the intermediate pituitary lobe of producing MSH 1.7. the cells of the parathyroid producing parathormone

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1.8. the parafollicular cells of thyroid producing calcitonin 2. Connexin signaling in cells producing glycoprotein hormones

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2.1. the myo-epithelioïd cells of kidney producing renin

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2.2. the thyrocytes of the thyroid follicles producing T3 and T4 2.3. the cytotrophoblast cells of placenta producing β-hCG 3. Connexin signaling in cells producing steroid hormones

3.2. the

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3.1. the cells of the adrenal cortex producing aldosterone, cortisol and sex hormones Sertoli

cells

of

testis

producing

inhibin

and

antimullerian

hormone

the Leydig cells of testis producing androgens 3.3. the theca cells of ovary producing estrogens 3.4. the luteal cells of ovary producing progesterone 3.5. the syncytiotrophoblast cells of placenta producing progesterone 4. Connexin signaling in the cells of the adrenal medulla producing catecholamine hormones 5. Connexin signaling in non secreting cells of endocrine glands 6. Connexins and endocrine disorders 6.1. the animal models 6.2. the human situation 6.3. the therapeutic perspectives 7. What we know and what we don’t Paolo Meda

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ACCEPTED MANUSCRIPT Acknowledgments

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References

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ACCEPTED MANUSCRIPT Introduction Several seminal studies that, close to 50 years ago, lead to the development of the gap junction-connexin-cell coupling field, have been made in secretory cells [1-10]. Since, a large

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body of evidence has documented that, at least in vertebrates, gap junctions, connexins and

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cell-to-cell coupling are obligatory features of all vital glands, even though the extent and type of theses junctions, proteins and mechanism vary in different gland types, specifically if they

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function in an exocrine rather than in a (neuro)endocrine manner [11-14]. The usual conservation of these variations in different animal species, is consistent with different functions and regulations of connexin signaling in specific glands, with regard to the biosynthesis and

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release of different secretory products, as well as of the differentiation, life and death of secretory cells. These findings have been critically evaluated in a series of excellent reviews by experts of different endocrine glands, which provide an in depth, state-of-the art coverage of the

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unique homeostatic systems that each gland represents [15-80]. The reader is further referred to this text for a broader summary and comparison of what has been learned in these different systems, and to references to both the early, semantic studies which opened the field in each

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gland, as well as to the more recent developments, if any. Here, special attention is given to the

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studies on the β-cells which produce insulin in the pancreatic islets, and on the myo-epithelioïd cells which produce renin in the kidneys, which have been a major focus of studies on the

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function of gap junction and connexin signaling in endocrine glands. These systems are central to the development of pathological alterations in animal models which closely mimic different forms of diabetes [81] and hypertension [82], two diseases of increasing worldwide incidence, which combine in the metabolic syndrome [83]. Genetic studies and analysis of human tissues

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have also recently provided the first evidence supporting the involvement of connexin signaling in the pathological alterations of these systems [37,46,54,67,69,80]. Here, we summarize this novel data, mention the critical information which is still lacking, and evaluate whether the current knowledge warrants the development of novel, connexin-targeted therapeutic approaches, that could become valuable additions to improve the present, somewhat limited and cumbersome therapeutic arsenal against common endocrine diseases.

1. Connexin signaling in cells producing peptide hormones 1.1.

the β-cells of pancreas producing insulin: the role of connexin-dependent signaling,

in these cells has been extensively reviewed [12-14,20,29,37,40,43,48,51-54,58,64,65,6870,72,80]. Under basal conditions, β-cells are electrically and metabolically coupled by numerous, small gap junctions only made of Cx36 [84-97] (Figs 1 and 2). During exposure to glucose, which is the major natural stimulus of this system, this coupling results in a rhythmic activity, which synchronizes the electrical and Ca2+ pulses of β-cells across each pancreatic islet Paolo Meda

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ACCEPTED MANUSCRIPT [88,90-92,98-101]. The transcription of the GJD2 gene, which codes for Cx36, and the size of gap junctions further correlate with the expression of the Ins gene and insulin content, which are increased in parallel by glucose, due to the transactivation by the transcription factor Beta2/NeuroD1 that binds to the promoter region of both the GJD2 and the Ins genes [87,102-

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104]. In contrast, the expression of Cx36 is reduced following a fat-enriched diet, which induces

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glucose intolerance [105-107].

In both cultures and mice, loss of Cx36 results in the full uncoupling of β-cells

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[88,89,93,98,101,108]. Mice null for Cx36 do not release insulin in the normal pulsatile fashion, due to loss of the normal intercellular synchronization of the calcium transients which are induced by glucose stimulation, and that trigger both the first and the second phases of insulin release [88,89,93,101,109] (Fig. 1). These islets also show increased basal release of the

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hormone, since β-cells can no more be inhibited by the hyperpolarizing currents generated in nearby cells [88,89,93,109]. These alterations cause an intolerance to post-prandial glucose

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levels [101]. In contrast, mice expressing about half the control levels of Cx36 remain normoglycemic, since about 30% of these levels are sufficient to preserve a normal insulin secretion in mice [101]. In vivo, loss of Cx36 further sensitizes β-cells to cytotoxic conditions,

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including to the pro-apoptotic effect of cytokines which concentrate in the islet environment at

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the beginning of type 1 diabetes [108,110,111] (Fig. 1). Conversely, mice over-expressing Cx36, or other connexin isoforms, appear fully protected against the same insults [108,111]. Pro-apoptotic cytokines and oxidized LDL particles reduce the expression of Cx36 via a ICER1-,

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AMPK- and NOS-dependent mechanisms [105,106,110,113]. Cx36 also plays a central role in the oxidative and ER stress induced by cytokines [106,110,113]. These effects enhance the mitochondria-dependent apoptosis of β-cells [106,108,110-113], indicating that Cx36 is also

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relevant for the survival of these cells, under pathophysiological conditions. Recently, we reported on a single nucleotide polymorphic variant (rs3743123) of the human GJD2 gene which does not modify the primary sequence of Cx36, still resulted in altered formation of gap junction plaques, and reduced coupling in vitro [114]. Transgenic mice expressing this variant only in β-cells, due to the control by an insulin promoter, consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines [114]. The study also revealed that rs3743123 GJD2 marginally associated to heterogeneous populations of diabetic patients, but reduced the expression of Cx36 in the pancreatic islets of hyperglycemic, but not normoglycaemic individuals [114]. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, possibly contributing to the pathogenesis of type 2 diabetes. We further documented that glibenclamide, a sulphonylurea widely used in the treatment of various forms of diabetes, and which enhances the formation of gap junctions, the expression of Cx36, and the coupling of β-cells [85,94,97,111,118], prevented the development of an autoimmune type 1-like diabetes in the strain of non obese diabetic (NOD) mice [111]. Thus, Paolo Meda

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ACCEPTED MANUSCRIPT and in marked contrast with the untreated littermates which were used as controls, the glibenclamide-treated mice remained normoglycaemic for weeks, due to the preservation of a sizable mass of Cx36-linked β-cells [111]. These effects occurred even though the glibenclamide-treated mice developed obvious signs of an autoimmune attack of the pancreatic islets (insulitis), alike the untreated control littermates [111]. The data imply that an early Cx36-

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targeted treatment may help protecting β-cells against the autoimmune attack, which triggers the development of type 1 diabetes.

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Collectively, these data show that the signaling dependent on Cx36 is hierarchically important in the multifactorial regulation of insulin dynamics, which is central to glycemic control. Still how the loss of Cx36 signaling alters insulin secretion and β-cell apoptosis remains incompletely unraveled. It is possible that this pivotal position results from the role of coupling which,

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compared to other forms of cell-to-cell communication, can equilibrate the intrinsically heterogeneous features of individual β-cells, and ensure their increased secretory recruitment

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during stimulation [37,99,100,116-118] This implies that, compared to other mechanisms of cellto-cell communication, connexin-dependent coupling may be particularly advantageous in tissues made of heterogeneous cells. Several lines of evidence indicate that this is the case for

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pancreatic islets, in which β-cells differ in a number of structural, biochemical and functionally

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respects, including in terms of the biosynthesis, storage and release of insulin [119-127]. At least some of these observations cannot be attributed to a different environment within different regions of the islets, inasmuch as marked differences in insulin secretion are also readily

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observed in vitro. Thus, individual β-cells simultaneously exposed to conditions of maximal stimulation by glucose or by other non metabolizable secretagogues, feature marked differences in the ability to release insulin [119-126], and retain this pattern for hours [127], in

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spite of similar electrophysiological changes indicating a comparable activation of the stimulussecretion coupling machinery [128]. This different behavior may be explained by a difference in the biosynthetic activity of β-cells [120,129], or in levels of some of their key proteins [130-133]. Under such conditions, coupling allows for the recruitment of increasing numbers of secretory cells with both cell aggregation and the degree of stimulation [99,101,119-122,134]. The underlying mechanism somehow involves connexins, inasmuch as drugs blocking junctional channels and antisense constructs interfering with connexin transcription, blocked the contactdependent recruitment of secretory β-cells during glucose stimulation [135]. The reason why βcells are functionally heterogeneous, and why their asynchronous functioning may be inappropriate should be experimentally tested. Lack of coupling prevents the sharing by β-cells of sparse ion channels that are critical for proper activation of the stimulus-secretion coupling [116-118]. Also, by inducing irregular oscillations of cytosolic Ca2+ and a basal, steady increase in the levels of this cation, β-cell uncoupling could also alter the expression of genes critical for secretion and/or control of β-cell apoptosis [136]. It is further possible that some deleterious effects of β-cell uncoupling may be accounted for by an altered cross-talk between Cx36 Paolo Meda

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ACCEPTED MANUSCRIPT signaling and the signaling provided by other molecules, including SUR1-Kir6.2, E-cadherin and ephrin A [136-139].

1.2. the α-, δ-, PP- and ε-cells of pancreas producing glucagon, somatostatin, pancreatic

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polypeptide and ghrelin: the circulating levels of somatostatin and glucagon regularly oscillate

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coincidentally (somatostatin) or antagonistically (glucagon) with the oscillations of insulin [37,48,51,54,69,140,141], implying the requirement for some forms of coordinating, hetero-

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cellular coupling [19,48,51,54,69]. Still, direct, unambiguous evidence that these cells express bona fide gap junction plaques [142], and connexin proteins is still lacking [37,48,51,54,69]. Also, tracer studies have not indicated an obvious connexin-dependent coupling between the β-

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and non β-cells of pancreatic islets, as well as between the latter α-, δ-, PP- and ε-cells [144146], except in vitro [143]. Therefore, it is possible that these islet cells communicate via

1.3.

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connexin-independent mechanisms [37,48,51,54,69].

the neurons of hypothalamus secreting GnRH, TRH, CRH, somatostatin, GHRH,

dopamine, vasopressin, and oxytocin: the connexin signaling of these secretory neurons

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has been recently reviewed [12,14,20,31,37,48, 63,66,72,79]. Some of these cells express

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Cx36 [147-151], whereas others express Cx43 [152,153], and still others Cx32 [154]. Coupling has been shown between most of these neurons, except those producing GnRH electrical

coupling

of

these

cells

ensures

the

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[147,149,150,151,153,155-159].The

synchronization of fast diffusing Ca2+ waves which, in turn, are thought to account for the pulsatile release of several hormones, including GHRH, GnRH, TRH, oxytocin, and LHRH [147,149,150,151,153,155-159].Thus, the down regulation of connexin channels resulted in

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disorganized Ca2+ patterns, and in the loss of the pulsatile release of some of the hypothalamic hormones [160-162]. However, whether the electrical coupling of neurons is necessary and sufficient to contribute to the pulsatile secretion of the hypothalamic hormones in vivo remains to be definitively demonstrated, given that this secretion is also modulated by multiple paracrine influences [163], and by neuron-glial cell coupling [160].

1.4. the acidophil and basophil cells of the anterior pituitary producing GH, PRL, FSH, LH, TSH and ACTH: the connexin and coupling patterns of the endocrine cells of the anterior pituitary have been extensively reviewed [12-14,20,33,35,39,41,42,44,48,61,72]. GH- and PRLproducing cells are coupled by Cx43 and Cx26 channels, which achieve an intercellular synchronization of Ca2+ transients [164-167,169-171]. Even though seasonal changes in the expression of pituitary Cx43 have been associated to changes in prolactin secretion [170,171], direct experimental testing has not yet demonstrated that connexin signaling is physiologically relevant for the secretion of pituitary hormones in vivo. This is due to the complex cellular Paolo Meda

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ACCEPTED MANUSCRIPT composition of the pituitary, and to the multiple control mechanisms, including the coupling of the folliculo-stellate cells [168,172-174], which cross talk with those dependent on multiple connexin isoforms, to control the gland. A recent study documents that Cx36 sustains the synchronous activity of pituitary gonadotrope cells, thus ensuring a proper secretion of LH in

1.5.

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response to GnRH, and that estradiol modulates the expression of pituitary Cx36[175].

the B pinealocytes of pineal gland producing melatonin: these endocrine cells

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express Cx26 and Cx45, and are coupled [25,48,72]. Since such a coupling is increased in vivo by norepinephrine, it is plausible that connexin signaling may contribute to melatonin secretion [176]. Still, there is yet no direct evidence that this is the case in vivo, after either light or neural

1.6.

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stimulation.

the melanotroph cells of the intermediate pituitary lobe producing MSH: these cells

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express Cx43 [72] and, as yet, are not known to be coupled. Also unknown is the function connexin signaling could have in this pituitary lobe. the cells of the parathyroid producing parathormone: parathyroid cells express Cx26

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1.7.

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and Cx43 [12-14,72], and are presumed to be coupled by gap junction channels [15,72]. However, there is yet no published evidence that such a coupling may be relevant, if not

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required for parathormone secretion.

1.8. the parafollicular cells of thyroid producing calcitonin: these cells express Cx26 [72],

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still it is not yet known whether they are coupled, and whether connexin signaling is relevant for calcitonin secretion.

2. Connexin signaling in cells producing glycoprotein hormones 2.1. the myo-epithelioïd cells of kidney producing renin: the contribution of connexin signaling

in

this

system

has

been

extensively

reviewed

by

several

groups

[34,37,46,48,55,59,67,71,77,80]. The renin-producing cells of adult rodents are coupled by minute gap junctions made of Cx40 and Cx37, which also interconnect these cells to the nearby endothelial and smooth muscle cells of each juxtaglomerular apparatus [177-187] (Figs. 2 and 3). Under control conditions, this system ensures a basal renin release within the afferent arterioles of the kidney cortex, which result in a normal blood pressure, via the modulation of the renin-angiotensin system [34,37,46, 55,59,67,80]. Loss of Cx40 markedly increases the synthesis and release of renin [188-192], resulting in a sizable, chronic hypertension of mice (Fig. 3), and in a displacement of the myo-epithelioïd cells, several of which locate outside the Paolo Meda

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ACCEPTED MANUSCRIPT afferent arterioles [193]. The increased renin release results from the loss of the normal negative feedback mechanisms by the circulating levels of angiotensin II, the increased pressure within intra-renal vessel and the macula densa [193-197]. These studies have further shown that elevated levels of COX-2 and eNOS also contribute to the excessive renin secretion

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observed in the absence of Cx40 [193-197], and that these effects are kidney autonomous,

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inasmuch as they are also observed in mice nil for Cx40 only in the renin-producing cells [198]. In addition, the selective restoration of Cx40 in these cells reduces both renin secretion and

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hypertension [198]. In contrast, the endothelium-specific deletion of Cx40 does not alter the blood pressure of mice [191], and the restoration of Cx40 expression in the endothelial cells of mice lacking Cx40 fails to reduce their hypertension [199].

Parallel studies have also

documented that loss of Cx37 does not alter the renal expression of Cx40, Cx43, Cx45, and the

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control blood pressure of mice [200] (Fig. 3). In contrast, the replacement of Cx40 by Cx45 induces a modest hypertension, in spite of normal levels of circulating renin [201,202]. The data

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show the prominent role of Cx40 in the regulation of renin secretion, a tentative conclusion further supported by the observation of increased expression of Cx40 in the kidneys of control rats that were made chronically hypertensive by reducing the blood supply via clipping of a renal

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artery [203], a maneuver known to significantly raise the levels of circulating renin, as a result of the decreased perfusion of the clipped kidney [204], and indicate that this role can also be

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achieved by Cx45 [201]. In both cases, the increase in the circulating levels of renin increases the resistance of peripheral vessels, due to the sustained activation of the renin-angiotensin

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system [188,203]. Indeed, Cx40-null mice feature an altered vasomotion of small arterioles, and the blockade of either the angiotensin converting enzyme or of the angiotensin II receptor AT1 reduces their hypertension [188,191,205]. The endothelial cells of many arteries are coupled by

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Cx40 and Cx37 (Fig. 3). It has been recently documented that both Cx40 and Cx37 associate with eNOS, increasing the in vivo activity of the enzyme and, thus, the production of NO [206]. Loss of Cx40, decreases the activity of the enzyme, resulting in a reduced relaxation of the arteriolar smooth muscle cells that causes an increased vascular tone, contributing to enhance the renin-dependent hypertension [206]. Opposite effects were observed in a model of volumedependent hypertension [204]. The data indicate the involvement of connexin signaling in the control of vascular tone, under a variety of physiological and pathological conditions [207]. Collectively, these data provide compelling evidence that renin secretion is mostly controlled by Cx40 signaling, by a mechanism that is activated within the renin-producing cells themselves [198]. While this mechanism remains to be fully unraveled, available data already indicate that it alters the local production of NO, it affects the expression of the renin gene, the packaging of the hormone within secretory granules, the modulation of its release, and the positional information of the renin-producing cells within the kidney cortex [188-193,206]. This anomalous positioning raises the intriguing possibility that the changes in renin secretion observed after Cx40 loss may also result from the altered micro anatomical organization of the juxtaglomerular Paolo Meda

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ACCEPTED MANUSCRIPT apparatus. Renin-producing cells are normally recruited in increased functional numbers in response to hypotension, when they are found in multiple regions of the nephron [20,210]. Obviously, this homeostatic regulation is lost after loss of Cx40, since increased cell recruitment was observed in spite of a sizable hypertension [195]. In this case, the renin-producing cells

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also featured smaller and more regular secretory granules than controls, further suggesting a

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shift in their differentiation pattern, as usually observed when cells of the renin lineage are not in their native niche, within the media of the afferent arteriole [211]. However, the hypertension of

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Cx40-deficient mice cannot be solely accounted for by the perturbed renin production, as the segmental vasoconstriction and altered vasomotion of small arterioles which is induced in these mice by the normal activation of the angiotensin production resulting from the excessive renin levels, also plays a significant role [191,108]. These findings raise the intriguing possibility that,

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beside the direct effects of a Cx40 on key proteins of the renin-producing cells, the changes in renin biosynthesis and release observed after Cx40 loss may also have been contributed by the

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altered coupling between the renin-producing cells and the other cell types of the juxtaglomerular apparatus.

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2.2. the thyrocytes of the thyroid follicles producing T3 and T4 : Several papers have

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reviewed the status of connexin signaling in the follicular cells of thyroid [12,14,16,18,20,48,72]. These cells express Cx32, Cx26 and Cx43, and are coupled throughout each thyroid follicle [12,212-216 ]. Correlative evidence suggests that this coupling may be implicated in the

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production of thyroglobulin, and the secretion of T4 , as well as in the morphogenesis of thyroid follicles [214,217-220]. However, no obvious thyroid defect has yet been reported in mice null

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for a connexin gene [48,72,221].

2.3. the cytotrophoblast cells of placenta producing β-hCG: what is known about connexin signaling in this gland has been reviewed[17,22,23,61,73]. Cx43 is found between the cells of both the cytotrophoblast and syncytiotrophoblast [222-224]. Impairing Cx43 signaling decreases the fusion of cytotrophoblast cells, as well as the expression of the β-hCG gene, and the secretion of this hormone [225,226]. More recently, it has been documented that the trophoblast also expresses Cx31 and Cx31.1, and that inactivation of either gene differently alters placental structure, resulting in partial embryonic loss at mid gestation [227]. Whether this effect involves an altered endocrine function of the placenta has not yet been investigated.

3. Connexin signaling in cells producing steroid hormones 3.1. the cells of the adrenal cortex producing aldosterone, cortisol and sex hormones: the connexin signaling of the adrenal cortex has been repeatedly reviewed [12,14,20,28,72,78]. The mineralocorticoid-, glucocorticoid- and sex hormone-producing cells of the adrenal cortex are all Paolo Meda

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ACCEPTED MANUSCRIPT coupled by Cx43 channels [12,228-231]. Correlative observations have documented the potential role of this connexin in the control of both the ACTH-induced secretion of cortisol, and the growth of the steroid-producing cells [232-236]. However, evidence has not yet been

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published, which could validate these effects in vivo.

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3.2. the Sertoli cells of testis producing inhibin and antimullerian hormone: Multiple reviews have summarized the connexin signaling between Sertoli cells, and between these cells

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and selected germ cells [12,21,24,32,36,38,48,50,56,62,64,76]. Sertoli cells are coupled via Cx43 channels [12,24,237-241]. Pharmacological and in vivo studies on genetically modified mice show that this coupling is involved in the control of spermatogenesis, and Sertoli cell

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growth [237-241]. Several other connexins (Cx26, Cx32, Cx33, Cx36, Cx45, Cx46 and Cx50) have later been also reported in the testis. Loss of Cx46 is associated with an increase in germ cell apoptosis and loss of the integrity of the blood-testis barrier [50,56,62,64,76]. Recently, it

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was reported that IL1-1α, a cytokine which disrupts the blood-testis barrier, increased the expression of Cx43, still decreased Sertoli cell coupling [242]. Conversely, 2-hydroxyflutamide, a metabolite of the anti-androgen flutamide, decreased Cx43 expression [243]. If these studies

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clearly establish a relevant and highly modulable role of connexin signaling in different functions

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of Sertoli cells, they have not directly tested whether these functions include their endocrine

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secretion.

3.3. the Leydig cells of testis producing androgens: Cx43, as well as Cx36 and Cx45, also couples the Leydig cells [12, 74,76,244-247]. Previous reviews have summarized the somewhat contradictory

findings

about

the

effects

of

Cx43

signaling

in

androgenesis

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[12,14,21,32,38,50,74,76]. The in vivo loss of Cx43 does not alter steroidogenesis or the development and differentiation of Leydig cells [244-247]. In contrast, loss of Cx43 from Sertoli cells results in Leydig cell hyperplasia, suggesting a connexin-dependent interaction between these two cell types [247]. In the absence of Cx43, Leydig cells were still coupled, suggesting that other connexins contribute to their gap junctions [247], and/or were induced to compensate the loss of Cx43. Thus, which connexin isoform is essential for androgenesis remains to be fully elucidated, specifically in vivo.

3.4. the theca cells of ovary producing estrogens: Expert publications have reviewed the multiple functions of connexin-dependent signaling in the endocrine cells of the ovary [12,14,26,48,73-76]. Theca cells of secondary follicles are connected by Cx43 and Cx37 [248250,253,254]. In vitro and in vivo experiments have documented that Cx43 contributes, with multiple paracrine signals [261], to the proper control of folliculogenesis and oocyte maturation [248-252,255-263,266], whereas Cx37, which connects granulosa cells of the cumulus Paolo Meda

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ACCEPTED MANUSCRIPT oophorus, hinders the meiotic competence of oocytes [249,264,265]. Whether and how these effects are related to an altered estrogen production and release has not been directly investigated.

connexin

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3.5. the luteal cells of ovary producing progesterone: The participation of

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signaling during the luteal phase of the ovarian cycle has been expertly summarized [12,14,26,48,73-76]. Cells of corporea lutea are connected by Cx43 [267,268,270,274]. In vitro,

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the pharmacological interference with junctional channels and Cx43 expression modulates progesterone secretion [268-272]. Still Cx43-null mice have normal gestations and litter sizes, suggesting that the connexin effect is not sufficient to impact on gestation, and/or that the loss

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of Cx43 can be compensated by some other mechanism. In vivo, mice lacking Cx37 develop abnormal corpora lutea [249,273].

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3.6. the syncytiotrophoblast cells of placenta producing progesterone: little is known about connexin signaling in this gland [17,22,23,61,73]. Cx43 connects the cells of both the cytotrophoblast and syncytiotrophoblast and interference with Cx43 signaling decreases the

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fusion of cytotrophoblast cells [222-226]. Whether this effect impacts on the production of

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placental progesterone has not yet been directly investigated.

hormones.

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4. Connexin signaling in the cells of the adrenal medulla producing catecholamine

Several publications have excellently reviewed the involvement of connexin signaling in the

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release of epinephrine and norepinephrine [12,14,20,27,30,45,48,49,72]. The chromaffin cells of the adrenal medulla are joined by Cx36 in all animal species and, in some species, also by Cx43 and Cx50 [275-278]. The resulting coupling mediates the cell-to-cell spreading of the Ca2+ transients driven by action potentials [275], which results in an amplification of the secretion of both epinephrine and norepinephrine, after the synaptic activation of individual cells [275,276]. The coupling of chromaffin cells is modest under basal conditions, but increases in stressed animals due to the up-regulation of Cx36 and Cx43 [277-280], and, under both conditions, appears modulated by cholinergic influences [281]. A study on anaesthetized mice has provided evidence that connexin signaling contributes to the control of catecholamine secretion in vivo [282]. However, no publication has yet investigated this question in animals models deprived or overexpressing selected connexin species.

5.

Connexin signaling in non secreting cells of endocrine glands

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ACCEPTED MANUSCRIPT Most tissues targeted by hormones are at a sizable distance from the gland that produces the endocrine products, usually preventing the establishment of connexin-dependent signaling between secretory and non secretory cells. However, connexins may be involved in the crosstalk between these two cell types when these are in close contact. This is the case, for example,

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of the juxtaglomerular apparatus, the endocrine regions of the renal nephrons, which produce

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renin (section 2.1.). The renin-producing cells of the afferent arteriole, which are central players of this apparatus, share Cx40 channels both with companion cells and adjacent endothelial

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cells, and the latter cells are further connected to the smooth muscle cells of the afferent arterioles [34,37,46,48,55,59,67,71,77,80]. Heterozygous transgenic mice in which the coding region of Cx43 is replaced by that of Cx32 in about 50% of the alleles, show a normal distribution of Cx43, and become hypertensive as a result of increased plasma renin levels

NU

[283]. In contrast, homozygous littermates, in which Cx32 had replaced Cx43 in all alleles, retained a normal blood pressure and control levels of circulating renin [283], providing the first

MA

support for a mechanism, whereby altered connexin signaling between endothelial cells modifies the functioning of the renin-secreting cells (Fig. 3). Also, in the pituitary, the non endocrine folliculo-stellate cells cross-talk with close by endocrine cells, by establishing gap

D

junctions that allow for a rapid propagation of the waves of cytosolic calcium across the entire gland [173]. This arrangement provides for an efficient mechanism to orchestrate the function of

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endocrine cell types that are scattered throughout the anterior pituitary. A comparable role has been proposed for the sustentacular cells of the adrenal medulla, which functionally extend the

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communication network of the endocrine chromaffin cells, and for the glial cells of neurosecretory regions of the central nervous system [20,27,30,31,37,45,48,49,60,63,66,72,79].

6.1.

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6. Connexins and endocrine disorders the animal models: many lines of transgenic mice, featuring a general or cell-specific

loss of a single connexin isoform, have been reported to show changes in selected endocrine functions, that mick specific alterations observed in major human diseases [e.g. 3032,34,37,38,43,45,46,48-51,54,57,58,59,64-67,69,72,73,74,76,77-80,111-114,175,206]. Fewer studies have reported analogous alterations in animals over-expressing a specific connexin, or in which the native isoform of the protein has been replaced by isoform [29,37,48,67,108,283]. When documented by independent groups, the available data are tantalizing, inasmuch as they strongly support the idea that connexin signaling may contribute both to the early pathogenesis of several common endocrine disorders, and to their long term maintenance. Still, in most of these cases, it remains unclear what is the specific effect of the connexin signaling, and, specifically, whether it has a causal effect on the initiation and development of a model diseases, or whether it is only one of the pleiotropic consequences of most pathological states, possibly participating to the development of their chronic complications. Paolo Meda

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ACCEPTED MANUSCRIPT In the case of pancreatic β-cells, mice invalidated for Gjd2, which codes for mCx36, feature several alterations in β-cell function, including loss of the synchronous, regular oscillations of free cytosolic calcium which are induced by glucose stimulation, loss of the consequent insulin oscillations, increased basal release of insulin, and failure to increase the insulin output in the

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presence of post-prandial concentrations of glucose [88,89,101,108,109,112,113]. As a result,

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at least a fraction of the Cx36-null mice develop glucose intolerance [101]. These alterations are reminiscent of those observed in humans affected by early pre-diabetes (loss of insulin

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oscillations) and, later, overt type 2 diabetes (all the other secretion changes). Analogous alterations have been observed after the β-cell specific invalidation of Gjd2, but not after the over-expression of Cx36 under control of a fragment of the insulin gene promoter [108], showing that the effect is pancreas autonomous, and directly, likely solely, on β-cells. Also, a

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single nucleotide polymorphism in the coding sequence of human GJD2 results in a post-natal reduction of Cx36 expression, which correlates with a partial loss of β-cells and glucose

MA

tolerance [114]. Conversely, conditions elevating the circulating levels of glucose and fatty acids and inducing glucose intolerance, obesity, peripheral insulin resistance, hyper-insulinemia, and increased beta cell mass of the animals, feature decreased Cx36 expression and β-cell

D

coupling, resulting in a reduced glucose-induced insulin secretion [106,113]. These alterations recall those featured by most patients affected by type 2 diabetes [81] and metabolic syndrome

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[82]. Mice invalidated for Gjd2 also show increased sensitivity to the pro-apoptotic Th1 cytokines, resulting in a sizable loss of pancreatic β-cells, whereas the over-expression of Cx36

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protects against both effects [108], either by enhancing β-cell resistance and/or by improving their intrinsic DNA repairing mechanisms. Conversely, Th1 cytokines decrease Cx36 expression [106-108,110,111,113]. These alterations are reminiscent of those observed in humans at the

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onset of type 1 diabetes. Strikingly, the administration of a sulphonylurea which promotes the in vivo expression of Cx36 and the coupling of β-cells, fully protects these cells from the spontaneous type 1 diabetes which normally develops with age in the mice of the non obese diabetes (NOD) strain, in spite of an obvious autoimmune attack of the pancreas [111]. In the case of the renin-producing cells of kidneys, the invalidation of the Gja5 gene, coding for Cx40, results in a marked increase in renin secretion, which results in a chronic hypertension of the transgenic mice [180,189,190,192,197]. These alterations are also observed following the renin cell-specific loss of Cx40, and are reduced after the restoration of the protein solely in this cell type, showing the predominance of this connexin in the kidneys [198]. Still, the hypertension of Cx40-deficient mice is also partly accounted for by peripheral vascular effects, which result from the renin-dependent activation of the renin-angiotensin system [179,180,191,207,208]. The Cx40 coupling of endothelial cells and the Cx43 coupling of the smooth muscle cells of peripheral vessels, specifically of resistance arterioles, both play a significant role in these effects. The scenario best fitting the available data posits that the alterations initiate in the endothelial cells where Cx40 and Cx37 control the expression and function of the eNOS Paolo Meda

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ACCEPTED MANUSCRIPT enzyme, which leads to a reduced production of NO [206]. Via either a gap junction or a paracrine pathway, this second messenger can no more sufficiently relax the adjacent smooth muscle cells, thereby impairing the proper adaptation of resistance vessels to the hemodynamic changes induced by the increased blood pressure [37,46,55,59,67,71,206,207]. Remarkably,

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both the renin and the vascular changes, which have been documented by independent groups,

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mimic the major clinical signs of most patients affected by chronic hypertension [82]. In the context of the so-called metabolic syndrome [83], type 2 diabetes is associated to lipid

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disorders and hypertension, leading to chronic, devastating complications of various organs, including the eye, the kidneys, the hearth, and most vessels. The connexin and the coupling patterns of these organs are altered by the increased levels of circulating glucose and fatty acids which characterize the human metabolic syndrome [105-107]. The data suggest an

maintenance

of

both

micro-

and

macro-angiopathies

[37,43,46,48,51,54,

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55,59,64,65,67,69,71,80].

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implication of several connexins, notably Cx40, Cx37 and Cx43, in the development and/or

Analogous homologies between the phenotype of mice genetically modified to invalidate the expression of Cx43, and the signs of some human diseases, have been reported in other

D

endocrine systems [20-25,26-28,30-32,35,36,38,39,45,48-50,56,57,60,62,63,66,73-76,78,79,].

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These studies have documented changes on both hormonal secretion and on the proliferation and death of the cognate secretory cells. Still, in several of these studies, a direct experimental

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testing is still lacking to document whether a cell-specific restoration of the connexin signaling can actually restore a proper endocrine function in vivo, making most of the published evidence at best correlative.

the human situation: The animal observations summarized above provide compelling

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6.2.

evidence that connexins significantly contribute to the in vivo control of multiple endocrine cell functions. Specifically, the finding that the phenotype of mice featuring cell-specific alterations of connexin signaling closely mimics the signs of human diseases [20-25,26-28,30-32,3539,43,45,46,48-51,54-57,59,60,62-67,69,71,73-76,78-80], and that the patterns of connexins and coupling are usually similar in most endocrine glands of humans and laboratory rodents [12,20,23,37,48,54,69,72], suggests that the signaling dependent on these proteins may be relevant in the pathogenesis of common endocrine diseases. For obvious ethical, safety and methodological reasons, this tantalizing possibility cannot be clinically validated in humans by the direct experimental testing which is feasible in laboratory animals. Thus, this possibility is supported by circumstantial evidence. In the endocrine field, this evidence is still quite modest, and has so far been derived from either genetic analysis of connexin gene mutations and polymorphisms in small, selected populations of individuals [37,80,114,284-289], or from immunological analysis of connexin proteins in a limited number of rather heterogeneous Paolo Meda

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ACCEPTED MANUSCRIPT pathological samples [17,18,23,56,71,73-77,290-294]. As yet, the essential proof of concept that a connexin-targeted pharmacological, molecular biology and/or cell therapy could correct a defect in connexin expression and/or function, thereby improving the signs of an endocrine disease has not been provided.

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In the case of diseases characterized by hyperglycemia, the GJD2 gene which codes for human

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Cx36, is located on the 14q region of chromosome 15, which is a susceptibility locus for type 2 diabetes and the diabetic syndrome [37,48,54,69,81,83]. A single nucleotide polymorphism

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(SNP rs3743123) in the coding sequence of GJD2, which is synonymous, i.e. does not alter the primary sequence of Cx36, marginally associates to large, highly heterogeneous populations of diabetic patients, still is unusually frequent in those type 2 diabetics that show the largest reduction in glucose-induced insulin secretion [114]. Expression of quantitative trait loci (eQTL)

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further shows that the pancreatic islets of these hyperglycemic individuals expressed less hCx36 transcripts than hyperglycemic individuals who did not carry SNP rs3743123 [114]. The

MA

data are consistent with those obtained after expression of the variant Cx36 in transgenic mice devoid of mCx36, which also featured a post-natal reduction of islet Cx36 levels. [114] In these mice, this reduction associates with a loss of the β-cell mass which was sufficient to result in the

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hyperglycemia of selected lines [114]. Altered expression of the Cx36 transcript has also been

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reported in genome wide scans of type I diabetics [37,48,54,69,284,285], consistent with the altered expression of the connexin in a model (NOD mice) of spontaneous, auto-immune diabetes, which is considered a valuable model of the human disease [111]. In this model, the

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administration of a sulphonylurea (glibenclamide in the EU, glyburide in the USA) which is widely used for the treatment of multiple forms of human diabetes, and which promotes the in vivo expression of Cx36 and the coupling of β-cells in rodent islets [37,48,54,69], fully protects

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NOD mice against the apoptosis induced by the autoimmune attack [111]. In the case of diseases characterized by hypertension, two polymorphisms (-44AA/+71GG) have been reported within the promoter region of the GJA5 gene, which codes for human Cx40 gene, and another (1019C/T) in the GJA4 gene, which codes for Cx37, that marginally associate

with

increased

risk

of

hypertension

in

selected

human

populations

[37,46,59,286,287]. Two other polymorphisms of Cx40 and Cx43 have been further associated with vessel alterations which are often associated with a chronic increase in blood pressure [288,289]. The data are consistent with the changes in blood pressure observed in rodents expressing the variant Cx40, as well as with the ex vivo data showing that this mutations reduce the permeability of the junctional Cx40 channels [286,287]. Altered expression and function of other connexins, notably of Cx26, Cx40 and Cx43, have been described in pathological samples of other human endocrines, specifically in samples of transformed glands [16,18,21,23,27,28,30,36,56,57,62,73,75,76,77,78,290-296]. The data suggest a participation of connexin signaling also in the complex, multiple steps regulation of cell proliferation, mobility and adhesion, which are deregulated during transformation and metastasis, as well as in the Paolo Meda

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ACCEPTED MANUSCRIPT vascular changes that are commonly associated with tumoral development [206,207,296]. However, whether these changes are causal in the tumoral transformation of endocrine cells, or are merely one of the many changes that such a transformation induces remains to be

T

adequately shown.

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6.3. The therapeutic perspectives: It should be obvious from the previous sections that connexins play a key, probably obligatory, if not essential role in the proper physiological

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functions of most vertebrate endocrine glands. The available data also provide the first clues that an altered connexin signaling may contribute to the abnormal multi-cellular and multi-organ interactions that characterize endocrine disorders. Therefore, the question arises whether we

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could take advantage of connexin biology to develop innovative therapeutic approaches to endocrine diseases [29,37,51,54,297-299]. Presently, the available hormonal replacement treatments for such diseases are, at best, individually cumbersome, not free of secondary

MA

effects and complications, and impose a serious burden on both patients and medical economy. A connexin-targeted approach could involve drugs and molecular biology constructs to modulate in vivo the expression and function of selected connexin isoforms, as well as newly

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engineered hormone-producing cells expressing adequate levels of native connexins for a

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proper integration into the cognate dysfunctional organs (Fig. 4). The difficulties of the endeavor are obvious and multiple, since the ideal goal would be to achieve a sufficient specificity of the

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therapeutic tools to target specific connexins in selected cell types, and to raise or decrease connexin signaling at levels adequate to properly modulate specific cell functions. Advancing towards such goals will require the development of methods whereby to rapidly screen, in a connexin sensible way, many types of compounds and cells [300]. Preliminary experimental

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studies have provided the proof of concept that connexin-targeted treatments are efficient in laboratory rodents, and rare others suggests that they may be translatable to the human clinic, opening the exciting perspective that the therapeutic goals mentioned above may not remain but a dream. In the case of diabetes, a sulphonylurea (glibenclamide in the EU, glyburide in the USA) which is already widely used in the treatment of various forms of the human disease, because it stimulates insulin release from glucose-insensitive β-cells, also promotes the assembly of Cx36 channels and improves β-cell coupling [13,20,29,37,48,51,54,69,80]. In the NOD mouse model, this drug fully protects against the spontaneous development of an auto-immune form of type 1 diabetes, in spite of the persistence of a sizable immune attack of the pancreas [111]. In view of the experimental data in other in vitro and ex vivo models, the molecule is expected to promote insulin secretion while decreasing β-cell apoptosis [13,20,29,37,48,51,54,69,80,108,110] (Fig.4). Since the expression of Cx36 is down-regulated by the key metabolic enzyme AMPK [110], it is plausible that these two favorable effects of Cx36 signaling could be further potentiated by combining the administration of the sulphonylurea, to enhance Cx36 production, Paolo Meda

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ACCEPTED MANUSCRIPT with drugs inhibiting AMPK, to block the inhibitory effect of the enzyme on both Cx36 and insulin secretion, as well as its stimulatory effect on β-cell apoptosis. Cell therapies, in which surrogate stem, progenitor or induced pluripotent stem cells are engineered for the in vivo replacement of endocrine cells, e.g. β-cells in the case of diabetes, are also contemplated but presently face serious drawbacks. Most of the cells that are the basis of many such trials do not express the

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connexins found in the cognate adult tissues [301-304], i.e. Cx36 for islet β-cells. This defect

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could well account for the insufficient maturation of a β-cell-like phenotype [102,103,305], as

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well as for their limited functional integration into a coherent endocrine network, limiting the effects of the cell replacement. Favoring a rapid establishment of the cell interactions mediated by connexins between the transplanted cells, and these cells and the host environment, by providing the surrogate cells with an adequate connexin pattern prior to transplantation, which is

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certainly feasible with available molecular and cell biology methods, should promote their in vivo function in recipient organs (Fig. 4).

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In the case of hypertension, the available data imply that preventing the loss of Cx40 should favor the prevention of the disease, if not allowing for the correction of the established alteration in blood pressure [37,46,55,59,67] (Fig.4). Awaiting the identification of drugs acting on this

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connexin with some specificity, molecular biology tools, which can effectively modulate Cx40

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expression and signaling [296-298], may be envisaged. The difficulty here will be to determine the right connexin dosage, inasmuch as an excess of the protein may favor an inadequate

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angiogenesis and accelerate the growth of transformed tissues [296]. Treatments have also been contemplated in the case of other endocrine systems [56,57,62,73,75,76,77,78,296-299,306], specifically in the case of their tumoral transformation. Mimetic peptides, miRNAs, sense and antisense constructs can be designed to selectively

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interfere with the production and function of specific connexins (Fig.4), in order to inhibit both the growth and angiogenesis of tumors [296-299,307-309], including in some clinical conditions [309]. Again, the connexin targeting agents would be expected to also potentiate the effects of existing treatments (e.g. by cytotoxic drugs), by favoring the delivery of the drug within tumors, via their beneficial effects on the structure of the newly formed vessels [296,309]. In this context, the therapy would be further expected to benefit of the persistence of functional gap junction channels, to favor the spreading of the treatment to many cells of the targeted organ. Clinical trials have begun in which retroviral vectors are used to express the herpes simplex virus thymidine kinase in tumoral cells. The transduced cells are then killed by ganciclovir, a guanosine analogue which, after phosphorylation by the viral enzyme, blocks cell proliferation by incorporating into nascent DNAs [310,311]. Even though, only a minority of the transformed cells can usually be induced to express the viral kinase, sizable areas of the tumor become sensitive to ganciclovir. If the mechanism of this "bystander effect" is still debated, the finding that the gancyclovir metabolite is transferred between infected and non infected cells via gap junctional channels, indicate a likely role of connexin signaling [311]. An analogous, beneficial Paolo Meda

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ACCEPTED MANUSCRIPT effect is “channel sharing”, whereby ionic fluxes into only a limited proportion of cells are sufficient to correct the defective functions of a much larger cell population [312,313]. For example, less than 10% of the cells lacking the cystic fibrosis conductance regulator protein (CFTR) need to be corrected, by CFTR transduction, to restore a normal fluid transport of intact

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epithelial sheets [313], given that connexin channels are permeable to chloride anions [37].

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Thus, there are several therapeutic windows to envisage the success of a connexin-targeted

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therapy.

7. What we know and what we don’t

It is now established that connexin play a relevant role in the multifactorial signaling network

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which has developed with evolution to ensure the safe, acute and long term control of vital endocrine secretions, under most physiological conditions. Specifically, connexin signaling appears physiologically necessary for the proper regulation of hormone biosynthesis and

MA

release, as well as for the modulation of endocrine cell growth and apoptosis. This signaling is multifaceted, as it involves a number of different connexins, and operates in quite different gland systems, that concur to fine control multiple vertebrate functions. The versatility and the fine,

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cell-specific modulation of the underlying mechanisms is remarkable. Recent observations

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further suggest that a dysregulation of this sophisticated signaling could contribute to some of the most frequent endocrine disorders of the human clinic. This possibility calls for further

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investigations on the molecular organization of the underlying signaling, the cellular mechanisms it controls, and the relationships between the connexin-dependent signaling and other pathways that concur to regulate the function of both endocrine and target cells. This knowledge is a prerequisite to evaluate the patho-physiological role of connexins in several

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endocrine disorders. In turn, this knowledge will provide critical information about the potential usefulness of a connexin-targeted strategy in the development of innovative therapies of these disorders.

With regard to the molecular mechanism whereby connexins affect the functions of endocrine cells, future studies should identify the endogenous signals which permeate connexin channels and act as second messengers. The task is complicated by the fact that many of the endogenous signal molecules and metabolites which permeate connexin channels are also important players for most types of endocrine secretions [13,20,37,48,54]. Also, it is conceivable that some of the biological effects of connexins may not imply the establishment of functional gap junction channels. Some connexons become inserted in non junctional domains of the cell membrane, i.e. at sites in which this membrane faces the extra-cellular medium. In this location, individual connexons may form the so called “hemi-channels”, which do not pair with the connexons of an adjacent cell [314-316]. These structures allow for the leakage of cytosolic molecules, notably ATP and glutamate, into the extra-cellular medium, and may permit the Paolo Meda

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ACCEPTED MANUSCRIPT reverse uptake into cells of large extra-cellular, membrane-impermeant tracers [316-321]. These fluxes are abolished by drugs blocking gap junction channels [322-325]. There is now undisputed evidence that several connexins, including the endocrine prominent Cx43 [319,321,326,327], Cx36 [328,329] and Cx40 [330,331] may form functional, i.e. conductive and

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permeant “hemi-channels”. However, it has proven difficult to unambiguously demonstrate the presence of functionally open and unpaired connexons made by these proteins under

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physiologically-relevant conditions [316,332], for example in the β-cells of pancreatic islets

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[333,334]. These considerations raise the intriguing possibility that these structures may be more frequent under pathophysiological conditions, than in normal tissues [334-337]. In contrast, a physiological relevance is not disputed for the non junctional channels made by pannexins, the family of three membrane proteins that share with connexins a similar

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membrane topography, and a comparable permeability to both current-carrying ions and larger, still low molecular weight membrane-impermeant molecules, but which differ from connexins by

MA

their primary amino acid sequence, and the presence of glycosylated moieties on the second extracellular loop [316,317,320,332]. Given that pannexin channels are often hardly distinguishable from connexin “hemi-channels” in native tissues, and that most endocrine cells express

one

or

several

pannexin

isoforms

in

addition

to

connexins

D

may

[80,316,317,326,320,332,334], it is still unclear whether any connexin “hemi-channel” operates

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in endocrine glands under physiologically relevant conditions and, if so, what could be its specific functional role. As it has been the case for the establishment of the junctional roles of

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different connexins in a few of the endocrine systems reviewed above, a definitive validation of any role of non junctional channels awaits in vivo studies, under physiologically relevant conditions, by different independent groups. Undoubtedly, the question is complex, since we do

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not have specific tools to selectively and reversibly turn off specific channels, in order to distinguish what may be due to connexins and to pannexins, and what may depends on gap junctional versus non junctional channels. Thus, drugs have but a limited selectivity, usually only dependent on their doses, for connexin and pannexin channels [80,332,338,339]. Specific iRNAs and miRNAs may help addressing this issue would a full, unambiguous effect be achievable on the channels of a given type which, so far, is hardly achievable. Similar limitations plague the use of specific antibodies and mimetic peptides that, in theory, should help differentiating the effects of junctional and non junctional connexin channels. Experiments testing the deletion or the over-expression of individual connexins, have also revealed cell effects that cannot be easily accounted for by alterations in the function of either gap junctional channels or non junctional “hemi-channels”[340-344]. Furthermore, deletion of a single connexin isoform has sometimes resulted in severe in vivo phenotypes, that could be ascribed to alterations in biochemical pathways, hitherto unsuspected to be linked to connexin signaling [345-347]. These data, indicating that connexins may signal cells by mechanisms that do not depend on changes of membrane conductance and permeability, have opened the Paolo Meda

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ACCEPTED MANUSCRIPT search for alternative mechanisms. For example, transcriptome analysis of brain has revealed that many genes are altered after loss of Cx36, implying that the protein may tightly, and coordinately control the expression of genes previously thought to have no obvious relationship [345-351]. The apparently selective and coordinated effect of different connexins on the

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genome, with the expression of a number of genes being up- or down-regulated in parallel,

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could certainly provide for an efficient amplification of the connexin effect, that could explain why the functional loss of a single connexin isoform can induce dramatic phenotypes, in spite of

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the persistence of the junctional coupling provided by other connexin isoforms [69,352-354]. This effect could be relevant in many endocrine cells which, as summarized in co-express multiple connexin species [37,46,51,59,80,sections1.3.-4.].

Several studies have further documented that several types of connexins co-localize with a

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variety of other membrane and cytosolic proteins, and may directly interact with them, within multi-protein complexes [206,355-359]. Most of these proteins are major components of other

MA

types of intercellular junctions, such as tight junctions and adherens junctions or are associated with these structures, still many others are prominent parts of the cytoskeleton [357,360,361]. Such a co-localization and functional interaction has now been also documented for connexin

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isoforms expressed by endocrine cells, including Cx43 [362-365], Cx40 [206] and Cx36 [138,139,360,361]. Loss of the coupling provided by Cx36 channels between the insulin-

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producing β-cells of pancreas, as a result of either cell dispersion, pharmacological blockade of the channels or inactivation of the Gjd2 gene, results in two apparently contradictory alterations

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of β-cell function, i.e. increased basal secretion and decreased glucose-stimulated release of the hormone [37,51,54,69]. This dual regulation has biological sense, since it provides low amounts of insulin between meals and much higher levels of insulin immediately after food

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intake. The underlying molecular mechanism likely involves β-cell signaling via an ephrin-A5 ligand/receptor mechanism, in which the forward signaling activated by the ephrin A receptor in the presence of low glucose levels inhibits basal insulin secretion, whereas the reverse signaling activated by the ephrin A ligand in the presence of high glucose concentrations stimulates insulin secretion [139]. The effects of the ephrin A mechanism were prevented after interference with Cx36 mRNA, raising the question of whether the ephrin-attributed effects, may actually be mediated by Cx36 changes and, conversely, whether effects so far attributed to Cx36 may have actually been due to altered ephrin signaling [37,51,54,69]. Collectively, these findings indicate that connexin signaling cross talks with the signaling provided by other cell communication mechanisms, stressing the necessity to now understand how such a cross-talk is hierarchically organized to ensure the proper integration of individual cells within a structurally and functionally coherent multicellular organism. With regard to the cellular mechanisms that are activated by connexins, future experiments should investigate which cell type is actually targeted in glands (e.g. the hypothalamus or the pituitary) which are made by different cell types that are tightly apposed. In this case, and due to Paolo Meda

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ACCEPTED MANUSCRIPT the almost consistent coexistence of a paracrine regulation, it is unclear which cell type is first targeted by, if not dependent on the initial connexin signaling. The situation is further complicated in those many endocrine glands whose secretory cells co-express multiple connexin isoforms. In only a very few cases (e.g. the renin-producing cells of kidneys) have

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these proteins be individually targeted and/or replaced by other proteins to establish their

IP

respective functional relevance for the system [34,37,46,48,55,59,67,71,77,80]. The results have shown that, if some basic functions may be performed by other types of residual

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connexins, other functions cannot. Thus, the respective roles of different connexins for different types of secretory cells remain largely unknown in most glands. Even in the case of a favorable system (the rodent pancreatic islets), in which a single connexin type (Cx36) forms functional gap junctions between a single type of endocrine cells (β-cells), a substantial cellular

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heterogeneity has been reported in vitro and in vivo. Thus, β-cells substantially and consistently differ in their ability to biosynthesize and secrete insulin [37,51,54,69,99]. The question

MA

therefore arise of which subsets of cells are initially targeted by the connexin signaling and why, and whether these cells are hierarchically more relevant than the nearby companion cells to drive a coherent function of the entire glands. Eventually, in at least one gland (the renin-

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producing cells of kidneys) we now know that a single connexin isoform (Cx40) drives in parallel effects that are mediated by gap junctional communication and others that depend on the

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production of a second messenger (NO) responsible for paracrine effects [37,46,80,198,206]. Most likely both the gap junction and the paracrine effects involve different cell types (the renin-

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producing cells, the endothelial cells, and the smooth muscle cells of the afferent arterioles). Again, the question is to determine which of these cells depend on the gap junctional communication for their full integration in the gland function. The question is also of relevance

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for several other endocrine glands (e.g. the hypothalamus, the pituitary and the adrenal medulla) in which non endocrine glial, stellate and sustentacular cells may be coupled by connexins to the endocrine cell types, which could help to diffuse and amplify the connexin signaling over sizable distances [12-14,20,33,35,39,41,42,44,48,61,72]. With regard to the possible patho-physiological role of connexin signaling, further studies are needed to determine whether qualitative and/or quantitative alterations in connexin expression/function participate in a causal or consequential manner to the altered structural organization and/or function of endocrine glands, specifically in animal models mimicking clinical human situations. Conversely, the observations made in such models should be validated by a parallel assessment of the alterations in the function of endocrine cells in models featuring a general or cell-specific deletion of individual connexins. Any such alteration should then be shown to be reversed after reestablishment of the connexin expression, under control of either the cognate, physiological promoter and/or after over-expression under control of an irrelevant, still cell-specific promoter. As yet, such a comprehensive approach has been applied to only a couple of endocrine systems [12-14,20,29,34,37,40,43,46,48,51-55,58,59,64,65,67Paolo Meda

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ACCEPTED MANUSCRIPT 72,77,80]. Eventually, it is now time to initiate expression studies on connexins in patients affected by endocrine disorders, at least by taking advantage of the genetic and molecular biology approaches that are now available [86,284-289]. This research should also investigate the acquired and environmental conditions which are thought to play a causal or at least

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favoring role in the initiation, maintenance and complications of endocrine disorders. So far, the

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effects of such conditions on connexin signaling have been unfrequently investigated [29,54]. This information is expected to provide important clues as to whether means to specifically

SC R

interact with connexin signaling represent an avenue worth further exploration for correcting the structural and functional alterations of endocrine glands. This may involve both genetic approaches to correct defects in the expression of the proteins, and the development of a connexin-targeted pharmacology. The unavailability of molecules with a sufficient selectivity to

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act on specific connexins is presently a major limitation in the field. The identification of better compounds requires the prior development of safe, easy to use and automatic methods for the

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high-throughput screening of candidate molecules [300]. The acid test of the value of connexins as therapeutic tools would obviously be the documentation that a change in connexin signaling can prevent/block the in vivo alterations characteristic of an endocrine disease. A single paper

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reporting that this may not be an inaccessible dream, at least in animal models, has been

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published, using a FDA-approved drug which is largely used in the diabetes clinic, and targets Cx36 [80,111]. Comparable studies should investigate other endocrine conditions of clinical relevance. In some of these disorders, a cell replacement therapy may be the approach of

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choice for a forthcoming and definitive treatment. This therapy implies that the surrogate, hormone-producing cells to be transplanted in the patients are able to functionally interact with the native, resident companion and supporting cells of the gland, in order to ensure an hormone

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secretion commensurate with the requirements of the host organism. The studies on laboratory rodents strongly imply that proper connexin signaling is as an obligatory requirement to achieve such integration. Still, if increasing data indicate that most of the human stem, progenitor and induced pluripotent stem cells modified for cell therapy do not express native patterns of connexins [301-304], little effort as yet been made in the endocrine field to test whether an adequate connexin expression could be instrumental to improve the so far limited beneficial effects of cell therapy [111,305]. Given the variety of specific connexin properties, and the many endocrine functions which appear selectively modulated by connexin signaling, not to mention the large number of hormones, each affecting a variety of target cells with an exquisite sensitivity and specificity, these challenges are formidable. If somewhat overwhelming, these challenges are quite exciting from a scientific standpoint. They certainly are also timely to progress in our understanding of several endocrine disorders whose pathogeny remains, at best, obscure. We can now expect that this understanding will be instrumental for the development of innovative therapies of those endocrine disorders, which have nowadays gained an exploding prevalence, worldwide. Paolo Meda

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ACCEPTED MANUSCRIPT Acknowledgments I thank the collaborators mentioned in the references. The work of my team has been continuously supported by grants from the Swiss National Science Foundation, the Juvenile

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Diabetes Research Foundation, and the European Union.

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[352] De Zeeuw CI, Chorev E, Devor A, Manor Y, Van Der Giessen RS, De Jeu MT, Hoogenraad CC, Bijman J, Ruigrok TJ, French P, Jaarsma D, Kistler WM, Meier C, PetraschParwez E, Dermietzel R, Sohl G, Gueldenagel M, Willecke K, Yarom Y. Deformation of network connectivity in the inferior olive of connexin 36-deficient mice is compensated by morphological and electrophysiological changes at the single neuron level. J Neurosci (2003) 23: 4700–4711. [353] Goodenough DA. The crystalline lens. A system networked by gap junctional intercellular communication. J Cell Biol (1992) 3: 49–58. [354]

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Koekkoek SK, Hoogenraad CC, Hamers FP, Gueldenagel M, Sohl G, Willecke K, De Zeeuw CI. Analysis of Cx36 knockout does not support tenet that olivary gap junctions are required for complex spike synchronization and normal motor performance. Ann NY Acad Sci (2002) 978: 391–404. [355] Hervé JC, Derangeon M, Sarrouilhe D, Giepmans BN, Bourmeyster N. Gap junctional channels are parts of multiprotein complexes. Biochim Biophys Acta (2012) 1818: 1844-1865. Paolo Meda

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connexins: emerging roles. Biochim Biophys Acta. (2004) 1662: 22-41. [357] Olk S, Zoidl G, Dermietzel R. Connexins, cell motility, and the cytoskeleton. Cell Motil Cytoskeleton (2009) 66: 1000-1016.

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[362] Butkevich E, Hülsmann S, Wenzel D, Shirao T, Duden R, Majoul I. Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton. Curr Biol (2004) 14: 650–658.

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[364] Laing JG, Chou BC, Steinberg TH. ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells. J Cell Sci (2005) 118: 2167–2176. [365] Shaw RM, Fay AJ, Puthenveedu MA, von Zastrow M, Jan YN, Jan LY. Microtubule plusend-tracking proteins target gap junctions directly from the cell interior to adherens junctions.

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Cell (2007) 128: 547–560.

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ACCEPTED MANUSCRIPT Legends Fig. 1. The β-cells of pancreatic islets. Top) The main cells of the endocrine pancreas, which store insulin granules (red), are coupled (yellow) by Cx36 channels (light green), and also

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express Panx2 (light blue) and Panx1 channels (blue). Under control conditions, β-cells have a

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low basal secretion of insulin and significantly increase this secretion during glucose stimulation, which induces a calcium-dependent, pulsatile release of the hormone, resulting in

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normoglycemia. Middle left) Loss of Cx36 increases basal insulin secretion, and reduces glucose-stimulated insulin release, resulting in glucose intolerance and hyperglycemia. These changes are associated with increased cytokine-induced β-cell apoptosis. Middle right)

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Analogous changes are observed in the presence of the rs3743123 variant of human Cx36. Bottom) Loss of Panx2 is also associated with loss of glucose-stimulated insulin release and

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with increased β-cell apoptosis.

Fig. 2. Different gap junctions couple the insulin- and the renin-producing cells. Top) Electron microscopy of freeze-fracture replicas shows several small gap junction plaques (black

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arrows) associated to tight junction fibrils (white arrows) at an interface between pancreatic β-

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cells (left panel). In comparison, the gap junction plaques found between adjacent reninproducing cells are less numerous, and are not associated to tight junctions (right panel).

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Bottom) Cryostat thin sections of pancreatic islets (left panel) and kidney cortex (right panel) were immunostained using antibodies to Cx36 and Cx40, respectively, which were revealed by protein A-coated gold nanoparticles. In both cases, high magnification views reveal the close apposition of the cell membranes typical of gap junctions, along a much narrowed intercellular

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space (asterisks). At these sites, pancreatic β-cells concentrate Cx36, whereas renal myoepithelioïd cells concentrate Cx40. β-cells are identified by insulin-containing secretory granules (arrows in left panel). Myo-epithelioïd cells are identified by large bundles of cytoplasmic myofilaments (arrows in right panel). Bar, 270 and 450 nm in top and bottom images, respectively

Fig. 3. The renin-producing cells of kidney. Top) The main, myo-epithelioïd cell type (RPCs) of the juxta-glomerular apparatus of kidneys, which store renin granules (red), are coupled (yellow) by Cx40 (green) and Cx37 channels (light green), and also express Panx3 (purple). The nearby endothelial cells (ECs) are also coupled by Cx40 and Cx37 channels. The adjacent smooth muscle cells (SMCs) are coupled by Cx45 (brown) and Cx43 channels (light brown). Middle left) Loss of Cx40 markedly increases renin biosynthesis and secretion, resulting in persistent hypertension. Middle right) Analogous changes are observed in the presence of the

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ACCEPTED MANUSCRIPT A96S variant of human Cx40 (dark green). Bottom) Loss of Cx37 does not significantly alter renin secretion and blood pressure.

Fig. 4. The translational perspectives. The available data indicate that the signaling provided

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by Cx36 improves (solid arrows) insulin secretion while inhibiting (flat end lines) β-cell

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apoptosis, whereas that provided by Cx40 inhibits the secretion of renin by the myo-epithelioïd cells of kidneys. An implication of these finding is that the pharmacological or molecular biology

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targeting of the signaling provided by these two connexin species could be (dotted lines) beneficial to restrict/inhibit different forms of diabetes and hypertension, possibly contributing to mitigate the resulting metabolic syndrome. Such a strategy is largely supported by in vitro and

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animal studies. However, its translational feasibility into the human clinic remains largely

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Geneva, December 16, 2016

Highlights

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In all endocrine glands, connexin channels couple the hormone-producing cells Connexins and coupling changes often correlate with hormone secretion changes Cx36 signaling modulates insulin secretion and the survival of pancreatic β-cells Loss of Cx36 causes β-cell alterations characteristics of type 1 and type 2 diabetes A synonymous SNP decreases Cx36 expression in hyperglycemic patients Cx40 signaling modulates renin secretion and epitheloid cell position in the kidneys Loss of Cx40 induces renin-dependent hypertension Cx40 mutations associate with some forms of human hypertension

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