T J Martin, J M Moseley and E D Williams. St Vincent's ... (Requests for offprints should be addressed to T J Martin) ...... the periosteum in young bones (Lee et al.
Parathyroid hormone-related protein: hormone and cytokine T
J Martin, J M Moseley and E D Williams
St Vincent's Institute of Medical Research,
(Requests for
offprints
Melbourne,
Victoria 3065, Australia
should be addressed to T J Martin)
Introduction
ancestral
The human PTHrP gene is complex than the PTH gene, with nine considerably exons that allow for alternate splicing to generate three different isoforms of 139, 141, and 173 amino acids in an
relationship.
more
Features of hyperparathyroidism have long been associated with malignancy, and with the advent of sufficiently sensitive bioassays, parathyroid hormone (PTH)-like activity was recognised in extracts of tumours from patients suffering from humoral hypercalcaemia of malignancy (HHM) (Stewart et al. 1983). While these extracts exhibited actions on bone and kidney that were very similar to those of PTH, low or undetectable levels of immunoreactive PTH in patients' plasma and in the tumour extracts indicated that the substance was unique (Stewart et al. 1980, Rodan et al. 1983, Stewart et al. 1983, Strewler et al. 1983). Subsequently, parathyroid hormone-related protein (PTHrP) was purified, sequenced and cloned from a human lung cancer cell line derived from a patient with HHM (Moseley et al. 1987, Suva et al. 1987). This protein, homologous with PTH in the amino\x=req-\ terminal region, acts through a common PTH/PTHrP receptor (J\l=u"\ppneret al. 1991) to promote bone résorption and inhibit calcium excretion. PTHrP is now known to be expressed in many normal tissues, including neuro¬ endocrine tissues, normal keratinocytes, endothelial cells, smooth muscle, lactating mammary tissue, and bone (review: Moseley & Gillespie 1995, Philbrick et al. 1996). At some of these sites PTHrP action is well characterised. Examples are relaxation of vascular (Nickols et al. 1989, Ishikawa et al. 199Ab) and uterine al. 1991, Barn et al. 1992, Dalle et al. 1992, Paspaliaris et al. 1992) smooth muscle and transplacental calcium transport (Rodda et al. 1988, Abbas et al. 1989, Care et al. 1990). At other sites, such as skin, bone and secretory mammary gland, a role in growth and differen¬ tiation processes is indicated (Amizuka et al. 1994, Kaiser et al. 1994, Karaplis et al. 1994, Wysolmerski et al. 1995). The general conclusion currently is that PTHrP functions as a paracrine regulator in many normal tissues. Crass &
(Shew
Scarpace 1993,
et
PTHrP gene
The PTH and PTHrP genes are thought to have arisen from a common ancestral gene through a chromosomal duplication event. The human PTH and PTHrP genes are located on chromosomes 11 and 12 respectively, and the genes for several other closely related proteins have been demonstrated on these chromosomes, consistent with such
length (Fig. 1) (review: Gillespie & Martin 1994, Moseley & Gillespie 1995). The genes of rat, mouse and chicken are slightly less complex (Fig. 1). Three spatially distinct promoters, two TATA and one GC-rich region, are responsible for transcription of the human gene, and these appear to be differentially regu¬ lated (Mangin et al. 1990, Vasavada et al. 1993, Southby et al. 1995). The PTHrP gene contains nucleotide sequence motifs in common with members of the immediate-early response gene family, as well as other hallmark features which include induction by growth
cycloheximide and relatively short-lived (Gillespie & Martin 1994, Holt et al. 1994). Many factors can regulate PTHrP gene expression, by altering the rate of transcription and/or changing mRNA stability. Glucocorticoids have been shown to inhibit PTHrP gene expression in a variety of cell lines, including factors,
serum or
mRNA
human squamous carcinoma cells (Kasono 1991, Kasono et al. 1991, Glatz et al. 199A), rat Leydig tumour cells (Liu et al. 1993), human cell lines showing features of neuro¬ endocrine cells (Ikeda et al. 1989, Lu et al. 1989) and human primary mammary epithelial cells (Sebag et al. 1994). Treatment with 1,25-dihydroxyvitamin D3 has been shown to down-regulate PTHrP gene expression in keratinocytes (Henderson et al. 1991, Kremer et al. 1991), human C-cell line (Ikeda et al. 1989), rat Leydig tumour cells (Liu et al. 1993) and amniotic fluid cells (Dvir et al. 1995). There are also reports that PTHrP expression was enhanced in a rat islet cell line (Streutker & Drucker 1991), keratinocytes (Henderson et al. 1991, Kremer et al. 1991) and a squamous carcinoma cell line (Merryman et al. 1993) following exposure to 1,25-dihydroxyvitamin D3. Thus the response to 1,25-dihydroxyvitamin D3 appears to be dependent on a variety of factors, including cell type and stage of differentiation, interactions with other cyto¬ kines, and the presence of serum. Treatment with 17ßoestradiol enhanced PTHrP mRNA expression in uterine cells, both in cell culture and in vivo (Thiede et al. 1991a, Paspaliaris et al. 1992, Casey et al. 1993), and in the rat pituitary and hypothalamus (Grasser et al. 1992), as well as in a rat pituitary cell line (GH-4C-1) (Holt et al. 1994), but had no effect on PTHrP gene expression in human
PTH III
human
-31H
84
PTHrP human I
II
-TATA
F
VI
III IV V
VIII
IX
i-+GC|->T/i
-36
1
III
rat
VII
r-»GCi-»TATA
139
173
141
V
IV
r-»GC|-»TATA -36
mouse
139
V
IV
III
r-XBCp+TATA -36
chicken
1
I
II
1
137
III
139
IV
- TATA
139 -36 1 Comparison of the organisation of the PTH and PTHrP genes. The coding regions (closed boxes), untranslated regions (open boxes), promoters (arrows) and splicing events (below each map) ate shown.
Figure
1
squamous carcinoma cell lines derived from the sub¬ mandibular gland or oesophagus (Kasono 1991). PTHrP mRNA was not regulated by 17ß-oestradiol treatment in Leydig tumour cells (Liu et al. 1993) despite previous demonstration of hormone responsiveness in this cell line (Veldhuis & Dufau 1993). As several growth factors and cytokines have been shown to modulate PTHrP production, and these sub¬ stances are often produced in the same tissues as PTHrP, it is likely that unique paracrine loops exist in each tissue to control PTHrP expression. In rat vascular smooth muscle cells, vasoconstrictors, such as angiotensin II, but not the vasodilator atrial natriuretic peptide, induce a rapid rise in
production (Piróla et al. 1993). PTHrP produced following such stimuli may then act locally in smooth muscle cells, possibly to oppose the vasoactive and/or growth-promoting effects of vasoconstrictor agents such as angiotensin II. Furthermore mechanical stretch increases PTHrP expression in vivo in bladder (Yamamoto et al. 1992), rat uterus (Daifotis et al. 1992) and aortic smooth muscle (Piróla et al. 1994). In vitro, the mechanical stimulus of rocking increases PTHrP expression in primary PTHrP
cultures of vascular smooth muscle cells, in a manner dependent on oscillation rate (Noda et al. 199A, Piróla et al. 1994). Thus increased PTHrP production following vaso¬ constriction or stretch may provide a mechanism to limit
these actions through the relaxant action of smooth muscle. In bone, PTHrP is produced osteoblasts, and its secretion is enhanced by transform¬
or reverse
PTHrP
on
by ing growth factor-ß (TGFß), epidermal growth factor, 1,25-dihydroxyvitamin D3 and glucocorticoids (Guenther et
al. 1995, Walsh
et
al. 1995, Suda
et
al.
1996).
higher levels of PTHrP exhibit a greater incidence of bone métastases when injected into mice (Guise & Mundy 1995). If this is correct it might follow that sufficiently sensitive assays for PTHrP in plasma could be used relatively early in the disease in patients with breast cancer to predict those at most risk for the development of bone métastases.
PTHrP
as a
hormone
PTHrP clearly functions as a hormone in those cancers in which it is produced in excess. Several plasma assays have been developed, identifying elevated levels in patients with the HHM syndrome, and incidents have been reported of PTHrP levels declining after tumour removal (Burtis et al. 1990, Sandhu et al. 1993) and of PTHrP gradients across a tumour bed (Sandhu et al. 1993). Furthermore, in an experimental model of HHM consist¬ ing of human cancers growing in athymic mice, treatment with neutralising antisera against PTHrP resulted in effec¬ tive treatment of the hypercalcaemia (Kukreja et al. 1988) and, indeed, was as effective as tumour removal in reversing the excess bone résorption seen histologically (Kukreja et al. 1990). There seems little doubt that PTHrP is the major mediator of hypercalcaemia in patients with the HHM syndrome, although it is possible that other bone resorbing cytokines could also contribute in some patients. Production of PTHrP is not confined to squa¬ mous cell cancers, but is also found in many other tumour types, commonly including kidney and breast cancers
(review: Moseley & Gillespie 1995). However, only a proportion of these patients become hypercalcaemic, and
the determining factors are the amount of PTHrP which reaches the circulation and the capacity of the normal homeostatic controls to regulate the calcium levels. It might be expected that in squamous cell carcinoma of the lung, for example, plasma levels of PTHrP would be elevated before any other biochemical effects are evident. Measurement of PTHrP with sufficiently sensitive plasma assays could be valuable in the early detection of lung cancer and in monitoring treatment of these and other cancers associated with humoral hypercalcaemia. In the case of breast cancer, another possible role arises for PTHrP, in addition to mediating the humoral hypercal¬ caemia noted in some of these patients (Grill et al. 1991). The high incidence of PTHrP expression by primary breast cancers (Southby et al. 1990), elevated plasma levels in 60% of those with hypercalcaemia and lytic métastases (Grill et al. 1991), and a higher incidence of PTHrP production in skeletal compared with non-skeletal mét¬ astases (Powell et al. 1991), have led to the hypothesis that PTHrP might help to endow breast cancers with the property of resorbing bone, thereby contributing to their facility for establishing and growing in bone as métastases (Powell et al. 1991). Recent work supports this notion with the demonstration that breast cancer cells expressing
Surprisingly, the influence of PTHrP on calcium metabolism in cancer is not confined to effects in solid tumours. It also extends to haematological malignancies, with clear evidence that PTHrP is the central factor in the hypercalcaemia which occurs very commonly in adult T-cell lymphoma/leukaemia, associated with HTLV-1 retrovirus (Honda et al. 1988, Watanabe et al. 1990, Johnston & Hammond 1992). Furthermore, PTHrP has been detected in other hypercalcaemia-associated haematological maligancies, such as multiple myeloma (Nakamura et al. 1992) and non-Hodgkins B-cell-type lymphoma (Nakamura et al. 1992, Wada et al. 1992). In healthy volunteers, circulating PTHrP levels are usually below the detection limits of current assays. How¬ ever, plasma levels of PTHrP have been detected in greater than 50% of a series of lactating mothers (Grill et al. 1992), and in the foetus (Thiebaud et al. 1993). PTHrP
a —
hormone in the foetus?
There is strong evidence that PTHrP is the major regulator of placental calcium transport acting by an endocrine pathway. Serum calcium is higher in the foetal than in the maternal circulation and the gradient is maintained by active transport of calcium across the placenta from the mother (Care et al. 1985). However, immunoreactive PTH levels are low in the foetus whilst PTH-like biologi¬ cal activity is high, suggesting the presence of another PTH-like substance (Care et al. 1985). Parathyroidectomy in the foetal lamb results in loss of the calcium gradient that exists between mother and foetus, implicating the para¬ thyroids as the source of the regulatory agent. Impairment of bone mineralisation also occurs in the foetuses of such experiments. Crude, partially purified or recombinant PTHrP, but not PTH nor maternal parathyroid extract containing no immunoreactive PTHrP, can restore the gradient (Rodda et al. 1988). Thus PTHrP appears to be the active component of the foetal parathyroid glands maintaining foetal calcium levels, and responsible for the depressed levels of foetal serum PTH. In support of this hypothesis, immunoreactive PTHrP is readily detectable in sheep foetal parathyroids from the time they form (Maclsaac et al. 1991), and also in early placenta, suggest¬ ing that the latter may be a source of PTHrP for calcium transport early in gestation. Some evidence indicates that the active domain in PTHrP which controls placental calcium transport lies between residues 67—86 (Care et al. 1990), but this remains to be fully established and the responsible receptor
has not yet been identified. The syncytiotrophoblasts are believed to be central in the transport of calcium to the foetus although the mechanisms are not well defined. However, the cytotrophoblasts which differentiate to form the syncytium are believed to be the calcium-sensing cells, and raising the extracellular calcium concentration has been shown to inhibit PTHrP release from these cells (Hellman et al. 1992). The mechanisms of placental calcium transport are not fully understood, but support for the role of PTHrP also comes from preliminary informa¬ tion from the PTHrP gene knockout mouse in which placental calcium transport is severely impaired (Kovacs et al. 1995). PTHrP
a
hormone in lactation?
-
The presence of PTHrP in pregnant and lactating breast tissue (Rakopoulos et al. 1992, Grone et al. 1994) and also in milk (Budayr et al. 1989, Khosla et al. 1990, Law et al. 1991, Ratcliffe et al. 1992, Kocabagli et al. 1995) indicates several possible roles for PTHrP, both paracrine and endocrine, in lactation and milk production. Extremely high levels of PTHrP are present in breast milk but it is not known whether it has a role in milk formation or in the transport of calcium into milk, or whether it serves as a nutrient to the neonate. Clinical studies have shown that lactation is associated with maternal bone loss and renal calcium retention, and in the light of current evidence PTHrP would be the most likely mediator (Kalkwarf & Specker 1995). Significant levels of PTHrP have been demonstrated in the circulation of lactating women (Grill et al. 1992, Bucht et al. 1995), where it may have an endocrine role to mobilise maternal bone calcium for milk production. In support of this hypothesis, significant bone loss is also associated 'with hyperprolactinaemia, which correlates with elevated PTHrP levels in non-lactating subjects (Stiegler et al. 1995). This correlation has also been observed in lactating women (Sowers et al. 1996). PTHrP expression in the rat mammary gland has been shown to be stimulated by suckling (Theide & Rodan 1988), and the demonstration of increased maternal renal excretion of cAMP and phos¬ phate in the rat in response to suckling (Yamamoto et al. 1991) indicates that during lactation PTHrP may reach the circulation to act subsequently on the kidney. PTHrP in lower vertebrates
The evolution of PTHrP and its role in submammalian species is of considerable interest. The widespread expres¬ sion of PTHrP and its role as a hormone in the foetus raised the question of whether it may function as a hormone in lower vertebrates. The early indications are that this may indeed be the case. In fish and the emerging frog, high levels of PTHrP expression have been demonstrated in the pituitary, hypothalamus, and kidney (Danks et al. 1993,
1997, Ingleton et al. 1995). Furthermore, a PTHrP-like protein has been detected in sea bream saccus vasculosus (Devlin et al. 1996) and eel corpuscles of Stannius (Chailleux et al. 1995). Substantial circulating levels of PTHrP can be detected in dogfish (Ingleton et al. 1995). The association of the pituitary with calcium regulation in fish (Pang & Pang 1986, Stiffler 1993) and the high levels of PTHrP expressed in the pituitary suggest that this may
be the source of circulating PTHrP. In addition, the PTH/PTHrP receptor has been cloned from amphibians (Bergwitz et al. 1996). Currently, work is under way aimed at determining the involvement of PTHrP in calcium homeostasis in lower vertebrates. PTHrP
as a
cytokine
Whereas PTHrP can be an endocrine effector in cancer, and perhaps also in lactation and in the foetus, there is now substantial evidence that it exerts paracrine functions in several foetal and adult tissues. The widespread expression of PTHrP in the developing embryo, particularly in epithelia at many locations and in adult skin, has supported the hypothesis that PTHrP is a cellular cytokine whose actions involve both cell growth and differentiation. In¬ deed, there is a growing body of evidence, particularly in epithelial cells, cancer cells, smooth muscle cells and in bone to support this notion, although its precise roles are not established. PTHrP and smooth muscle relaxation Relaxation of smooth muscle from PTH has been well documented
variety of tissues by (review: Mok et al.
a
1989/)); however the physiological relevance of these observations has been questioned (Cooper et al. 1991). The localisation of PTHrP mRNA and protein to the smooth muscle layer in these tissues, together with experiments demonstrating relaxation in response to PTHrP, has led to the hypothesis that locally produced PTHrP may act in an autocrine/paracrine fashion to modulate smooth muscle contractility. The relaxant activity of PTH and PTHrP is mediated by the PTH/PTHrP receptor, mRNA for which has been localised to smooth muscle cells in many organs (Urena et al. 1993). In the gastrointestinal tract, PTHrP mRNA has been detected in rat stomach, duodenum and colon (Cooper et al. 1991), and positive immunoreactivity for PTHrP has been demonstrated in the muscle layer of the stomach (Ito et al. 1994). Rat fundic strips were relaxed by PTHrP(l-34) (Mok et al. 1989a) and PTH/PTHrP receptor mRNA has also been demonstrated in the fundus wall, although these exper¬ iments did not discriminate between the mucosa and muscle of the stomach (Ito et al. 1994). Precontracted smooth muscle cells isolated from guinea pig ileum also relax when exposed to PTHrP(l-34), a response
attenuated
by
the PTH/PTHrP receptor
[Tyr34]-bPTH(7-34)NH2 (Botella et al.
1994).
antagonist
In the rat bladder, PTHrP mRNA levels have been shown to be proportional to the extent of bladder disten¬ sion. PTHrP(l-34) relaxed carbachol-induced contraction of bladder strips only when endogenous bladder PTHrP expression was low, that is when the bladder was kept empty (Yamamoto et al. 1992). This suggests that in the full bladder, endogenous PTHrP had desensitised the bladder smooth muscle to the relaxant effect of exogenous PTHrP. PTHrP is likely to be an important regulator of vascular tone and blood pressure, as was originally indicated by studies using PTH (review: Mok et al. 1989/>). Although production of PTHrP by the endothelium has recently been demonstrated (Ishikawa et al. 1994 ), the vascular endothelium is not required for exogenous PTHrP to produce vasodilatation (Nickols et al. 1989). However, it is possible that PTHrP may act as an endothelium-derived relaxing factor under normal physiological conditions. PTHrP mRNA (Hongo et al. 1991, Noda et al. 199A, Okano et al. 199A) and PTH/PTHrP receptor mRNA (Okano et al. 1994) have been demonstrated in aortic vascular smooth muscle cells and human coronary artery
(Nakayama et al. 1994), and PTHrP can induce relaxation a variety of arteries, including the rat aorta (Crass & Scarpace 1993, Ishikawa et al. 1994/)), rat femoral artery (Nyby et al. 1995), and in chicken shell gland vessels (Thiede et al. 1991 >). In addition, cultured vascular smooth muscle cells respond to PTHrP(l-34) with generation of cyclic AMP (Nyby et al. 1995), and in the aorta, PTHrP-induced cyclic AMP production has been in
correlated with decreased tension (Ishikawa et al. 1994/), 1995). However, in the rabbit kidney, it appears that nitric oxide synthase activation and generation of nitric oxide play an important role in the vasodilatory response to
(Simeoni al. 1994). The effect of PTHrP on blood vessels appears to vary with the vascular bed. The intravenous administration of PTHrP(l—34) led to reduced resistance and enhanced blood flow to heart and skin, and reduced renal resistance, but renal flow was not altered (Roca Cusachs et al. 1991). Conversely, PTHrP decreased blood flow and increased vascular resistance to the splanchnic organs. A similar differential effect on regional arterial systems has been previously demonstrated for PTH, although it appears that these differences may be species-specific (Mok et al. 1989/)). Thus locally produced PTHrP may play a role in regional blood flow regulation. PTHrP mRNA in blood vessel smooth muscle cells has been shown to be increased by vasoconstrictors (Hongo et al. 1991, Piróla et al. 1993) and mechanical distension (Noda et al. 1994, Piróla et al. 199A). These increases in PTHrP mRNA are followed by increased peptide pro¬ duction, and its secretion by aortic smooth muscle cells into the culture medium (Noda et al. 1994). In human PTHrP
coronary arteries, the intensity of PTHrP mRNA expres¬ sion by smooth muscle cell has been correlated with the degree of coronary artery stenosis. Thus, PTHrP may be involved in the paracrine control of vascular contrac¬ tility and/or smooth muscle differentiation and vascular
remodelling.
Desensitisation of the response of smooth muscle to exogenous PTHrP has been reported to occur in the smooth muscle of blood vessels (Okano et al. 199A, Nyby et al. 1995) and gut (Mok et al. 1989a), and it has been postulated that such desensitisation could account for the failure to detect hypotension in all patients with elevated PTHrP levels associated with humoral hypercalcaemia of
malignancy. Cardiovascular system
protein have been demonstrated in foetal and adult heart in several species (Moniz et al. 1990, Campos et al. 1991, Kramer et al. 1991, Moseley et al. 1991, Bui et al. 1993, Deftos et al. 1993, Burton et al. 1994). As well as the vasodilatory property of PTHrP (which results in decreased blood pressure in vivo (Nickols et al. 1989)), PTHrP(l-34) also has direct effects on the heart. In rat models PTHrP(l-34) increased heart rate both in vitro and in vivo (Nickols et al. 1989). A positive inotrophic effect of PTHrP(l-34) has also been postulated (Nickols et al. 1989), although there is some dispute about whether this is a direct effect or a consequence of vasodilation and increased heart rate (Ogino et al. 1995). The physiological role(s) of PTHrP in the heart remain to be determined, and may include modulation of cardiac output through vasodilatory and positive inotrophic actions, and/or action as growth factor during normal PTHrP mRNA and
development
or
pathological changes.
et
extra-embryonic tissues PTHrP is expressed in the uterus (Thiede et al. 1990), uterine cells (Casey et al. 1992), placenta and placental membranes (Abbas et al. 1990, Senior et al. 1991, Ferguson Uterus and
al. 1992, Bowden et al. 1994, Dunne et al. 1994, Dvir al. 1995, Wlodek et al. 1996), and placental cells (Hellman et al. 1992). Its expression is functionally regu¬ lated (Thiede et al. 1990, Paspaliaris et al. 1992, 1995, Williams et al. 1994), but once again its precise role at the many sites of its expression is uncertain. However, in addition to its demonstrated regulation of placental calcium transport (Rodda et al. 1988, Abbas et al. 1989, Care et al. 1990), it is likely to be involved in a number of uterine and placental functions. In the early stages of pregnancy, PTHrP mRNA is detectable in uterine epi¬ thelium at the site where implantation subsequently occurs (Beck et al. 1993). It is expressed also in giant trophoblast cells (Senior et al. 1991) and in decidual cells of both et
eí
pregnant and
pseudopregnant animals (Beck et al. 1993) and thus may be involved in growth and differentiation of these cells in response to pregnancy. The involvement of PTHrP as a paracrine regulator of uterine smooth muscle relaxation is demonstrated by PTHrP effects on uterine contractility (Shew et al. 1991, Barn et al. 1992, Dalle et al. 1992, Paspaliaris et al. 1992, Williams et al. 1994). Regulation of its expression by oestrogen (Thiede et al. 199Id, Paspaliaris et al. 1992), the oestrous cycle (Paspaliaris et al. 1992) and its temporary expression during pregnancy (Thiede et al. 1990, Williams et al. 1994) all point to specific, functional roles in the uterus. Oestrogen treatment has been found to en¬ hance the response of the non-pregnant uterus to PTHrP (Paspaliaris et al. 1992), and a recent study showed that PTHrP mRNA expression in the uterus was inhibited by dexamethasone, leading the authors to speculate that the high circulating levels of cortisol at term may be effective in promoting labour by reducing PTHrP and its relaxation effects on the uterus (Paspaliaris et al. 1995). Evidence for a specific and regulated role of PTHrP in the uterus during gestation comes from the observation of a temporal pattern in the relaxation response to PTHrP by longitudinal uterine muscle during pregnancy in the rat with maximal responses at times when oestrogen levels would be high. In contrast, the circular muscle did not respond at any stage during gestation (Williams et al. 1994). The inability of PTHrP to relax uterine muscle in the last stages of gestation does not support a direct role in the onset of parturition. It has been hypothesised that PTHrP may be involved in keeping the uterine muscle relaxed to accommodate the foetus during pregnancy. Thiede et al. (1990) demonstrated that expression of mRNA was dependent upon the presence of the foetus and that levels increased throughout pregnancy and decreased sharply after delivery. It seems likely, therefore, that the observed fall in PTHrP reflects the recontracted state of the uterine muscle, consistent with the observation in the bladder (Yamamoto et al. 1992), and that the level of expression is functionally related to contractility. The temporal expression of PTHrP in the endometrial glands and blood vessels (Williams et al. 1994) also supports roles in other regulated functions that might include uterine growth during pregnancy and the regulation of uterine and placental blood flow (Mandsager et al. 1993). PTHrP mRNA and protein have been detected in rat and human placenta in various cell types (Germain et al. 1992, Hellman et al. 1992, Emly et al. 1994 ). In addition, PTHrP is secreted by neoplastic cells of placental origin, including hydatidiform moles and choriocarcinomas in vitro (Deftos et al. 1994). The presence of PTH/PTHrP receptor mRNA has been demonstrated in rat placenta (Urena et al. 1993), and infusion of PTHrP(l-34) into isolated human placental lobules stimulates cyclic AMP production (Williams et al. 1991). Two further observa¬ tions lend support to the hypothesis that PTHrP is
involved in placental/uterine interactions and that its most likely role in the placenta and placental membranes is related to the growth and maintenance of the placenta itself during pregnancy. First, PTHrP production by cultured amniotic cells has been shown to be regulated by prolactin, human placental lactogen, insulin, insulin-like growth factor, and epidermal growth factor (Dvir et al. 1995); secondly, PTHrP has been shown to regulate epidermal growth factor receptor expression in cyto¬ trophoblast cultures (Alsat et al. 1993), an event associated with placental development. Although many functional studies remain to be completed, potential roles for PTHrP produced by the foetal membranes and placenta include transport of calcium across the placenta, accommodation of stretch of membranes, growth and differentiation of foetal and/or maternal tissues, and vasoregulation. The most likely source of the increased amniotic fluid PTHrP during gestation (Ferguson et al. 1992) is the amnion itself, since PTHrP mRNA expression is also highest at term and greater in the amnion than in choriodecidua or placenta (Ferguson et al. 1992, Bowden et al. 1994, Wlodek et al. 1996). In tissue from women with full-term pregnancies and not in labour, the concen¬ tration of N-terminal PTHrP has been found to be higher in amnion covering the placenta than in the reflected amnion covering the decidua parietalis (Bowden et al. 1994). Nevertheless, the concentration of N-terminal PTHrP in reflected amnion, the layer apposed to the uterus, was inversely related to the interval between rupture of the membranes and delivery. The observation that PTHrP levels in the amnion decrease after rupture of the foetal membranes has led to the proposal that PTHrP derived from the membranes may inhibit uterine contrac¬ tion, and that labour may occur following loss of this inhibition. Human foetal membranes have been shown to inhibit contractions of the rat uterus in vitro (Collins et al. 1993), so this tissue does appear to produce factors that can modulate uterine activity. Furthermore, primary cultures of human amniotic cells secrete PTHrP into the medium (Germain et al. 1992). Thus, while the physiological function(s) of amnion-derived PTHrP is currently un¬ known, the preliminary evidence suggests that it may play a role in the regulation of the onset of labour. It is also possible that it is a source of PTHrP ingested by the foetus, with a growth factor role in lung and/or gut development. Foetal growth and
development
expressed in the human and rat foetus (Campos al. 1991, Moseley et al. 1991, Burton et al. 1992, Dunne et al. 1994). These immunohistochemical studies have revealed a changing pattern of detectable protein, which is PTHrP is et
parallelled by changes in mRNA transcription, through¬ out gestation. Expression of both mRNA and peptide
occurred in endo-, meso- and ectodermal structures of the consistent with local production of the peptide in
foetus,
adult tissues. PTHrP immunoreactivity was identified in epithelia from many sources, including skin, bronchus, pancreas, pharynx, gut, stomach, and renal pelvis. Thyroid and parathyroid glands, which develop from epithelial origins, also stained positively for PTHrP, as did kidney collecting tubules, adrenal tissue, and skeletal and smooth muscle. These data support the hypothesis that PTHrP may function as a foetal growth and/or differentiation factor in many tissues. PTHrP immunoreactivity was also located in develop¬ ing long bones and calvaría, where it may have relevance in bone turnover during foetal development (Moseley et al. 1991). As discussed in the section 'PTHrP in bone\ mice homozygous for PTHrP gene disruption showed abnor¬ malities of endochondral bone development and distur¬ bances in cartilage growth, indicating that PTHrP plays an important role in normal skeletal development. In the mouse, PTHrP has been detected immunohistochemically in compacted morula stages, primarily in developing trophoectoderm cells, and in cells lining the blastocoelic cavity, which are likely to be primitive endoderm cells (van de Stolpe et al. 1993). Murine embryonic stem cells and F9 embryonal carcinoma cells show induction of PTHrP mRNA and protein, PTH/ PTHrP receptor mRNA and functional adenylate cyclasecoupled PTH/PTHrP receptors during differentiation to primitive and parietal endoderm-like cells (van de Stolpe et al. 1993). In F9 embryonal carcinoma cells, exogenous PTHrP(l-34) mimics dibutyryl cyclic AMP in triggering full differentiation of these cells towards a parietal endoderm-like phenotype (Chan et al. 1990, van de Stolpe et al. 1993). These results suggest that PTHrP, through the PTH/PTHrP receptor, may provide an autocrine/ paracrine mechanism to induce parietal endoderm differ¬ entiation. In rat embryonic tissues, the earliest time point that has been examined is day 8 of gestation, when PTHrP
demonstrated in trophoblastic cells but not in other embryonic and extra-embryonic tissues (Senior et al. 1991). Senior et al. (1991) suggested that PTHrP secreted by trophoblastic cells could act in a paracrine manner to maintain adjacent parietal yolk sac cells in a manner analogous to that described above for F9 embryonal carcinoma cells. The PTH/PTHrP receptor has been detected in many embryonic rat (Lee et al. 1993, 1995) and mouse mRNA
was
(Karperien et al. 199A) tissues, including intestine, lung, dermis, heart, kidney and bone, and the temporal and
spatial pattern of expression is similar to that of PTHrP. Thus the PTH/PTHrP receptor is present in tissues that also produce PTHrP. Notably, the receptor is usually detected in mesenchymal tissue adjacent to PTHrPproducing cells (Lee et al. 1995). Activation of the PTH/ PTHrP receptor is critical for foetal development in utero, as mice homozygous for gene deletion of this receptor die in mid to late gestation (see following section). Thus PTH/PTHrP receptor is clearly very important for foetal
development remains
to
be
in utero, although in fully elucidated.
precisely which
tissues
PTHrP in bone
PTH-like actions of PTHrP in bone were recognised in patients with humoral hypercalcaemia of malignancy long before its isolation and cloning. While PTHrP may have an endocrine effect on bone in the foetus and on maternal bone during lactation, most evidence now indicates a local paracrine role for PTHrP in the growth and differentiation of bone cells in the foetus and possibly also in adult life. Localisation of PTHrP in normal foetal bone (Moseley et al. 1991, Karmali et al. 1992), adult bone (Walsh et al. 1994) and cartilage cells (Tsukazaki et al. 1995) and the gross abnormalities of endochondral bone formation seen in mice homozygous for the disrupted PTHrP gene (Amizuka et al. 199A, Karaplis et al. 199A) now indicate the importance of PTHrP in bone development and poten¬ tially also in adult life. As a result of abnormalities in the growth and differentiation of chondrocytes in the genedisrupted animals, there is accelerated progression of differentiation resulting in shortened limbs (Amizuka et al. 1994). In contrast, but consistent with these observations, overexpression of the PTHrP gene in chondrocytes in vivo results in inhibition of the differentiation process and delayed osteogenesis (Henderson et al. 1995). It is note¬ worthy that the homozygous PTH/PTHrP receptor knockout mouse has a very similar phenotype to the PTHrP gene knockout although it is more lethal, death occurring in utero (Lanske et al. 1996). In contrast, the PTHrP gene knockout mice can survive several hours after birth (Karaplis et al. 1994). This observation alone indicates the possibility of other ligands for the PTH/ PTHrP receptor, either PTH itself or other as yet un¬ identified PTH-like substances. Of particular interest is the loss
recent can
by Amizuka et al. (1996) that bone detected in heterozygous mice with
observation
be
readily
PTHrP gene deficiency. In transformed bone cells, the amount of PTHrP secreted into the medium by a bone cancer cell line transfected with hPTHrP cDNA was related to the proliferation of the cells both in vitro and in vivo, following transplantation of the cells into nude mice (Pasquini et al. 1995). In these experiments, cells that were producing relatively high amounts of PTHrP in culture (compared
with the parent cell line) proliferated very slowly and when they were injected into nude mice no bone mét¬ astases were detected. When the effect of PTHrP on apoptosis of cultured chondrocytic cells was examined, transient expression of PTHrP containing the intact nucleolar targeting signal in the carboxy terminus delayed apoptosis induced by serum deprivation (Henderson et al.
1995).
Both PTHrP and PTH/PTHrP receptors are regulated by TGFß, which is highly expressed in foetal growth plate
Table 1 Reported effect of endogenous PTHrP on proliferation of mammary tissue, keratinocytes and bone cells. Stimulation (f) and inhibition (J.) of proliferation are shown by arrows. These data have been collated from studies in the literature, in which a variety of techniques have been used. These include the use of exogenous PTHrP" and PTH/PTHrP receptot antagonists'1, PTHrP antibodies', anti-sense technology11, PTHrP gene knockout1' and PTHrP overexpression systems' Cell line In vivo
Transformed
Primary
Tumorigenic
Cell type
Mammary
J.Wysolmerski
Keratinocyte
¿Holick
et a/. ( 1994b )a'b
et
al. (1995)
(1994a)1'
¿Ferrari
et
IHolick
et
ÎBenitez-Verguizas
al. (1992)
al. (1994a)a;
Kremer ei al. (1996)a
et al. (1992)d, (1994)d; Kremer et al. (1996)a;
¿Kaiser
Whitfield
et
¿/¿Henderson Osteoblast
al. (1996)a et al. (1991)a
IKanoeta/. (1991 )a; Li & Drucker (1993)'
Chondrocyte
¿Amizuka
et
al. (1994)e
î Lanske et al. (1996)ae; Loveys et al. (1993)a
and induces their differentiation (Jongen al. 1995, Tsukazaki et al. 1995). Reduction of the growth rate of RCB 2.2 cells (Li & Drucker 1993) and UMR 106 osteoblasts (Pasquini et al. 1995) transfected with the PTHrP gene is also consistent with PTHrP's growth inhibitory role, which had been indicated by studies in keratinocytes (Kaiser et al. 1992, 1994). Both PTHrP and PTH/PTHrP receptors have been identified in developing chondrocytes and osteoblasts of the periosteum in young bones (Lee et al. 1993, Iwamoto et al. 1994, Jongen et al. 1995, Tsukazaki et al. 1995), and there is some evidence that their levels of expression are regulated during differentiation of these cells and by growth factors (Lee et al. 1993, Guenther et al. 1995, Suda et al. 1995, Tsukazami et al. 1995). For example, retinoic acid treatment of pre-osteoblast UMR 201 cells, which induces a more mature phenotype, also results in decreased expression of PTHrP (Suda et al. 1995). The precise role of PTHrP in chondrocyte growth and differentiation is not yet fully understood. While it is clear that the actions of N-terminal regions of the molecule are mediated via the PTH/PTHrP receptor, the relative contribution of other parts of the molecule, which may act at intracellular targets, remains to be investigated. All of the foregoing evidence is strongly indicative of a paracrine function of PTHrP, and in particular that it might be concerned with the complex process of bone
chondrocytes et
elongation.
&
Esbrit (1994)aÄC; Bitch et al. (1995)a JLuparello et al. (1995)a
A most
exciting
recent
development
comes
from the observations that PTHrP appears to be a crucial molecule in the signalling pathway for cartilage differen¬ tiation, mediating the action of Indian Hedgehog protein (Vortkamp et al. 1996). Indian Hedgehog protein was first identified as playing an important role in delineating the
borders of Drosophila segments, and has since been found to play a key role in the formation of skeletal structure by cartilage cells. The protein regulates chondrocyte differ¬ entiation, and evidence indicates that it uses PTHrP as an essential signal, acting through the PTH/PTHrP receptor to achieve this (Lanske et al. 1996, Vortkamp et al. 1996). Elegant clinical confirmation of the view that PTHrP has an important paracrine role in bone growth and elongation comes from the report (Schipani et al. 1995) of mutations in the gene for the PTH/PTHrP receptor in patients with Jansen's metaphyseal chondrodysplasia. The mutations result in the receptor being in a persistently activated state, explaining the patients' hypercalcaemia and hypophosphataemia, which resembles that which occurs in patients with HHM. The short-limbed dwarfism in the patients with Jansen's metaphyseal chondrodysplasia reflects the important role emerging for PTHrP in bone growth and development.
of growth and differentiation In addition to acting as an agent involved in bone cell differentiation, there is increasing evidence that PTHrP can influence proliferation and differentiation of a variety of cell types in vitro and in vivo (Table 1; Adachi et al. 1990, Burton & Knight 1992, Emly et al. 199Ab, Iwamura et al. 1994, Li et al. 1996, Vasavada et al. 1996). This is Modulation
supported by the demonstration of the temporal and spatial expression of PTHrP and PTH/PTHrP receptor in many developing foetal tissues (Karperien et al. 1994, 1996). The proliferation and differentiation of keratinocytes in culture is modulated by PTHrP as follows. When endogenous PTHrP production was inhibited in an
established human keratinocyte cell line using antisense RNA technology, antisense-infected cells showed accel¬ erated growth indices (Kaiser et al. 1992). Interference with PTHrP production also inhibited expression of maturation-specific keratinocyte indices (Kaiser et al. 1994). These data indicate that endogenous PTHrP acts to inhibit growth and enhance differentiation in this keratinocyte model. Similarly, epidermal proliferation was inhibited by PTHrP(l-34) and increased by a PTH/ PTHrP receptor antagonist in vivo (Holick et al. 1994 ,/)). Furthermore, experiments that targeted overexpression of PTHrP to the skin of mice resulted in the failure to initiate follicle development or a delay in the initiation of follicles (Wysolmerski et al. 1995). It is likely that the effect of PTHrP may depend on the state of differentiation of the tissue (Whitfield el al. 1996), as PTHrP expression is reduced in confluent keratinocytes which express a more differentiated phenotype (Wysolmerski et al. 1994). In the mammary gland, overexpression of PTHrP by breast myoepithelial cells or local infusion with exogenous PTHrP(l—36) resulted in hypoplasia (Wysolmerski et al. 1995). PTHrP also inhibited mitogen-stimulated vascular smooth muscle cell proliferation (Piróla et al. 1993, Joño et al. 1994). Constitutive levels of immunoreactive PTHrP are low in normally cycling vascular smooth muscle cells (but not cancer cells) and accumulate during the latter stages of the cell cycle (Okano et al. 1995), suggesting a role for this protein in the process of smooth muscle cell division. Thus PTHrP appears to act as a growth inhibitor in a variety of tissues, probably through the PTH/PTHrP receptor. In contrast to these effects in non-malignant cells, in a variety of carcinoma cell lines endogenous PTHrP appears to have proliferative effects (Table 1). Whether this is due to tumour-specific responses and dysregulated production of PTHrP, associated with the cancerous state, or is a reflection of tissue-specific growth effects of endogenous PTHrP, remains to be determined. It is also possible that the nature of the exposure to PTHrP, such as intermittent or continuous exposure, affects the cellular response, as has been indicated in the studies of C-terminal PTHrP on keratinocyte growth (Whitfield et al. 1996). The growth of a human renal cell carcinoma cell line was almost totally inhibited by polyclonal PTHrP antiserum, and growth was significantly inhibited by a competitive PTH/PTHrP receptor antagonist (Burton et al. 1990). Similarly, trans¬ plantation of H-500 Leydig tumour cells into rats follow¬ ing transfection with full-length preproPTHrP mRNA in the antisense orientation reduced the tumour volume by more than 50% compared with control tumour cells (Rabbani et al. 1995). Thus in a wide variety of cell types there is growing evidence of a role for PTHrP in controlling proliferation, potentially by modulating proliferation and/or apoptosis. Furthermore, the presence of the nucleolar targeting sequence may be important in determining the effect of
the
endogenous form(s) of PTHrP on normal growth and development in each specific tissue and also under certain pathological conditions. It is not clear through what mechanism(s) PTHrP exerts effects on cell proliferation,
differentiation and apoptosis, but these may include con¬ trol of cell cycle progression and modulation of intracellular calcium levels. Although not fully understood, the contri¬ butions of the N-terminal, PTH-like region, of PTHrP to growth responses have been addressed. However, potential involvement of the nuclear processing sequences (Henderson et al. 1995) and C-terminal regions (Whitfield et al 1996) to growth, differentiation and apoptosis have also been raised, and will be the subject of much interest in the future. PTHrP in the central
nervous
system
PTHrP and PTH/PTHrP receptors have been demon¬ strated in brain (Weir et al. 1990, Fraser et al. 1993, Urena et al. 1993, Weaver et al. 1995, Eggenberger et al. 1996), cultured astrocytes (Hashimoto et al. 1994, Struckhoff & Turzynski 1995) and anterior pituitary (Weaver et al.
1995). Incubation of either N-terminal PTHrP, or PTH, with neuronal cultures induced a rapid rise in both cAMP and cytosolic free calcium (Fukayama et al. 1995). Homologous desensitisation of the cAMP response to both PTH and PTHrP was observed; however only PTH desensitised the calcium response (Fukayama et al. 1995). It is most likely of relevance that a second PTH receptor (PTH2) has been identified in brain and pancreas which is only activated by PTH (Usdin et al. 1995). The PTH2 receptor appears to couple to both the cAMP and intra¬ cellular calcium signalling pathways (Behar et al. 1996). The C-terminal PTHrP peptide PTHrP(107-139) has recently been shown to elicit a calcium but not a cAMP response, apparently in subpopulations of cells different from those in which a cAMP response was elicited by N-terminal peptides (Fukayama et al. 1995). Novel recep¬ tors for C-terminal PTHrP might, therefore, be expected in the brain, and it will be of considerable interest to ascertain the biological consequences of its action. While we know nothing of the physiological actions of PTHrP in the brain, we can speculate that it may be involved in neurotransduction, as intracerebroventricular injection of PTHrP increases dopamine specifically in the hypothalamus (Harvey et al. 1993). By inference from observations in other tissues, it is also expected that it will be involved in the regulation of blood flow and nerve growth and differentiation.
Summary acts as a hormone when it is produced in certain cancers, and perhaps also in lactating by women, the foetus, and lower vertebrates, it seems clear
While PTHrP excess
an important paracrine regulator of several functions. Its roles in smooth muscle relaxa¬ tissue-specific tion, placental calcium transport, and bone development are becoming firmly established. However, it is likely to be an important player in the control of cellular growth and differentiation, although much of our understanding of this role to date comes from indirect evidence. The next decade will be an exciting time in defining further both the hormonal and paracrine actions of PTHrP.
that PTHrP is
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