Imagine taking a pill to control your pain and, instead, the medication actually increases the pain you feel.
That may be the situation for patients who take opioids, but even more so for women, according to groundbreaking research by investigators at the University of Arizona College of Medicine – Tucson in the Department of Pharmacology.
The researchers identified a mechanism that explains why women may be more vulnerable than men to develop pain in general, as well as to develop pain from opioids specifically.
The cause is a neurohormone, prolactin, known largely for promoting lactation in expectant mothers in their final months of pregnancy and after childbirth.
Frank Porreca, PhD, associate department head, a professor of Pharmacology, anesthesiology, cancer biology and neuroscience at the college, and senior author on the study, notes it always has been understood that women experience some types of pain that occur without injury (known as “functional pain syndromes”) more than men.
The reasons for this never were clearly understood. A possible explanation the researchers explored was the differences in the cells and nerves that send pain signals to the brain in women and men.
The findings suggest new pain-management therapies targeting the prolactin system would greatly benefit women suffering from functional pain syndromes.
“Of all these female-prevalent pain disorders, migraines are among the most common, with about 35 million migraine patients in the United States, and three out of four of those are women.
In addition, in fibromyalgia patients, as many as nine out of 10 are women; for irritable bowel syndrome, three out of four are women.
When you add up all those women with pain – if you can normalize that – this would provide a huge and important impact on medical care,” Dr. Porreca says.
He points out many of these pain spells are intermittent and associated with triggering events. For instance, he and his colleagues found stress releases prolactin and unexpectedly promotes pain selectively in females.
“These triggering events can be wide-ranging. They can include things like alcohol, fatigue and sleep disruption. But stress is the most common trigger self-identified by patients. That’s where we started our studies – how does stress contribute to female-specific pain or female-selective pain?”
The cause is a neurohormone, prolactin, known largely for promoting lactation in expectant mothers in their final months of pregnancy and after childbirth.
Primary authors on the paper include: Yanxia Chen, a graduate student in Dr. Porreca’s lab; Aubin Moutal, PhD, a research assistant professor in the Department of Pharmacology, working in the lab of Rajesh Khanna, PhD, a UArizona professor of anesthesiology, pharmacology and neuroscience, who also is a co-author on the paper; and Edita Navratilova, PhD, an assistant professor of pharmacology.
Dr. Navratilova says dopamine D-2 receptor agonist drugs that limit prolactin release, such as cabergoline, commonly are used for other diseases, and are not addictive.
These drugs, possibly in conjunction with other classes of medications, may help treat those pain conditions in women more effectively without the addictive properties of opioids.
“If we could just reduce the proportion of women who have migraines to the same amount as in men, that would be quite revolutionary,” Dr. Navratilova says.
In addition, since publication of their findings, Dr. Porreca has been contacted by companies interested in investigating whether an antibody previously associated with breast cancer treatment might be able to be engineered as a therapy to guard against pain in women.
In recent years a renewed focus on sexual dimorphic mechanisms of pain has emerged. It is now widely recognized that many key mechanisms driving persistent pain differ between males and females in both animals and humans (Martin et al., 2019, Mogil et al., 2011, North et al., 2019, Sorge et al., 2011).
Although time course and magnitudes of nociceptive hypersensitivity for a variety of pain conditions are often similar in females and males, the mechanisms responsible for this hypersensitivity and degree of chronicity are sex dependent (Martin et al., 2019, Mogil et al., 2011, Rosen et al., 2017, Sorge et al., 2011, Sorge et al., 2015).
Gonadal hormones, for instance, are known to be key contributors to sex differences in a variety of physiological and pathophysiological processes (Karp et al., 2017, Morselli et al., 2017).
Human and animal studies of pain symptoms and severity have established correlations with the menstrual cycle, menopause, and alterations in gonadal hormone concentrations (Aloisi and Sorda, 2011, Houghton et al., 2002, LeResche et al., 2003, Slade et al., 2011, Traub and Ji, 2013).
Recent findings on sexual dimorphisms have demonstrated a role for spinal microglia in male-specific pain mechanisms (Sorge et al., 2011) and a T cell selective contribution to nociceptive transmission in females (Rosen et al., 2019, Sorge et al., 2015), although other investigators have described T cells to be involved in protection and resolution of pain (Krukowski et al., 2016, Laumet et al., 2019).
It is possible that a neuron-specific, sexually dimorphic pain mechanism also could be involved and mediated by gonadal hormone controlled signaling. A prime candidate for this potential mechanism is prolactin (PRL) and its receptor (Prlr), since responsiveness to PRL in a variety of cell types depends on sex, menstrual cycle phase, pregnancy status, and lactation (Belugin et al., 2013, Childs et al., 1999, Diogenes et al., 2006, Pi and Voogt, 2002).
PRL is involved in female-specific regulation of transient receptor potential (TRP) and other ligand-gated channels in sensory neurons (Diogenes et al., 2006, Liu et al., 2016, Patil et al., 2013b). Global ablation of PRL and Prlr leads to a substantial and female-selective reduction in postoperative and inflammatory heat hypersensitivity (Patil et al., 2013a, Patil et al., 2013b) and mechanical hypersensitivity, but the latter effect is observed in male and female mice (Patil et al., 2013a, Patil et al., 2013b).
These studies demonstrate a clear role for PRL-Prlr signaling in pain hypersensitivity after injury, but the cells mediating these effects and the mechanisms generating female-specific nociceptive responses remain unknown.
The central goals of the work described here were to gain insight into whether Prlr expression in sensory neurons drives female-specific nociceptive responses to PRL and to understand how these female-specific effects emerge.
We show that PRL signaling to Prlr expressed in sensory neurons at the level of peripheral and central terminals regulates female-specific hyperalgesia in several pain models. We also elucidate mechanisms responsible for PRL’s female-selective actions in the regulation of pain. Gonadal hormones regulate cellular phenotypes via classic genomic and transient non-genomic signaling pathways (Amandusson and Blomqvist, 2013, Kelly et al., 1976, Revankar et al., 2005).
However, surprisingly, our work points to a novel mechanism for sex-specific regulation of nociceptor plasticity that is dependent on selective and estrogen-dependent translation of Prlr mRNA in female DRG neurons. Overall, our work establishes sensory neuron participation of a major neuroendocrine hormone PRL in female-selective regulation of pain as well as a novel paradigm connecting sex- and gonadal hormone-dependent translational control that could be critical to understanding sexual dimorphism in many biological processes.
Studies in animals and humans demonstrate clear sex dimorphisms in mechanisms that control development of chronic pain (Dance, 2019), and the existing paradigm is that these dimorphisms are directly or indirectly regulated by gonadal hormones (Traub and Ji, 2013). A growing body of research suggests that an important mechanistic difference between development of chronic pain in male and female mice is that distinct immune cells are critical drivers, microglia in males (Paige et al., 2018, Sorge et al., 2011, Sorge et al., 2015, Taves et al., 2016) and T cells in females (Sorge et al., 2015).
The male-specific microglia effects are conserved for mice and rats (Mapplebeck et al., 2018) and can be conferred to females with testosterone treatment (Sorge et al., 2015). On the other hand, the T cell findings are controversial because T cells also play a critical role in pain resolution in some pain models (Krukowski et al., 2016, Laumet et al., 2019).
Several recent studies have also found sex differences in mRNA expression using RNA sequencing in whole human DRG and tibial nerve (North et al., 2019, Ray et al., 2019). The DRG transcriptomic work suggests that of the monocyte lineage in DRG and nerves may play a critical role in promoting neuropathic pain in males but to a lesser extent in females (North et al., 2019).
Sex differences in the tibial nerve transcriptomes, although not directly related to chronic pain since the tissues were from organ donors and not patients, found a clear signature for gonadal hormones in regulating transcriptomes in this tissue across the lifespan in females (Ray et al., 2019).
Collectively, these findings in rodents and humans support the classical viewpoint that gene regulation via gonadal hormone nuclear receptor-mediated transcription control mechanisms is a cornerstone of sex-dependent processes (Ormandy and Sutherland, 1993) including sex dimorphisms in pain (Rosen et al., 2017).
Our findings suggest a new twist on this paradigm wherein gonadal hormones could also regulate sensory neuron excitability via regulation of translation machinery or transcription of proteins belonging to the translation complex. As a key example, we demonstrated this mechanism for female-selective in vitro and in vivo PRL responsiveness in sensory neurons.
Previous studies showed gonadal hormones-dependent regulation of Prlr in non-neuronal cells (Furigo et al., 2014, Hu et al., 1996, Hu et al., 1998, Tanaka et al., 2005), but this is the first demonstration of such an effect in sensory neurons. Since Prlr does not have classical gonadal hormone-response element (Ormandy and Sutherland, 1993), Prlr mRNA expression is thought to be regulated by E2 utilizing alternative transcription binding sites, such as C/EBP, Sp3, and/or Sp1A (Dong et al., 2006, Goldhar et al., 2011).
Based on the literature, it could be extrapolated that Prlr mRNA should be predominantly expressed in female DRG neurons in an estrogen-dependent fashion. However, surprisingly, our data, which were generated by multiple, independent methods, show that Prlr-L and Prlr-S mRNA expression in sensory neuronal subtypes are not sex- or E2-dependent. Post-translational (i.e., phosphorylation, glycosylation) regulation is also unlikely, since prolonged E2 treatment (>6 h) is required for establishing PRL sensitivity in male neurons despite expression of Prlr mRNA.
Our data support the conclusion that translation regulation of Prlr function is critical for the observed dimorphisms in nociceptive processes during inflammatory pathological pain conditions. Accordingly, inhibition of cap-dependent translation almost entirely ablated PRL responsiveness in Prlr-cre+ female neurons and blocked the behavioral response to PRL in vivo.
In further support of this model, we showed that blockage cap-dependent translation eliminated E2-established PRL responsiveness in male DRG neurons. Moreover, we also observed robust expression of Prlr-cre+ sensory neurons and fibers in both male and female mice but found substantially higher Prlr protein expression in female rodent (rat and mice) spinal cord.
Based on these findings, we propose that sex- and E2-dependent translational regulation could be a novel mechanism for sexual dimorphism observed in many pain conditions (Fillingim et al., 2009). This is especially relevant considering that translation control mechanisms are already known to strongly contribute to modulation and sensitization of nociceptors but suggests that therapeutic targeting of these mechanisms may have additional benefits in females (Khoutorsky and Price, 2018, Megat and Price, 2018). Gonadal hormone-controlled translational regulation has been a subject of speculation but not studied in detail (Bronson et al., 2010, Ochnik et al., 2016).
This mechanism could be due to an increase of efficiency of translation by gonadal hormones and/or gonadal hormone-controlled additional mRNA transcription of proteins involved in translational machinery. Thus, translation regulation factors encoded by Eif2s3y and Eif2s3x genes exhibit strong sex dependency in mRNA expression in many types of neurons (Armoskus et al., 2014, Ray et al., 2019).
E2-driven translational control over the suppressor of cytokine signaling (SOCS) protein family has been proposed (Arbocco et al., 2016, Matthews et al., 2005) where E2 can affect the translational machinery via mTOR phosphorylation (Augusto et al., 2010) and regulation of Rheb signaling (Pochynyuk et al., 2006).
Translation can also be controlled by factors binding to mRNA. One of such factors is Musashi (Msi-1 and 2 genes), which binds specific sequences in the 3′ untranslated region (UTR) of mRNA and controls translation of proteins. It was shown that leptin can control translation of proteins by regulating Msi-1 expression (Odle et al., 2018).
These signaling pathways also play key roles in pain sensitization whereby they control the on-demand synthesis of new proteins that alter the excitability of nociceptors (Khoutorsky and Price, 2018, Moy et al., 2017).
A corollary of our work is that molecules involved in sex-dependent regulation of nociceptive pathways could be (1) induced by injury, (2) controlled by gonadal hormones, and (3) capable of regulating many other genes. PRL and its receptor Prlr fit these requirements. First, Prlr-mediated PRL effects are sex dependent in many tissues and cell types (Belugin et al., 2013, Ben-Jonathan et al., 2008, Patil et al., 2013a, Torner et al., 2001).
It is well documented that PRL responsiveness is closely controlled by E2 and to a lesser extent progesterone. Many clinical and preclinical studies also show that stress related to injury and inflammatory conditions trigger PRL release not only from the pituitary (Chernow et al., 1987, Noreng et al., 1987, Yardeni et al., 2007), but also from extra-pituitary tissues, such as cells in skin and in the spinal cord (BenJonathan et al., 1996, Patil et al., 2013a, Scotland et al., 2011).
Finally, Prlr activation could lead to epigenetic changes and transcription regulation of many genes via the STAT5 pathway (Ben-Jonathan et al., 2008, Bole-Feysot et al., 1998). This downstream transcriptional control may lead to additional diversification of nociceptor responses to injury since this pathway would not be induced in male neurons, which have very low levels of Prlr functional protein in most sensory neuronal types.
The preponderance of data on sex-specific mechanisms of chronic pain has focused on male rodents. To our knowledge, this is one of the first demonstrations of a female-specific inflammatory pain mechanism acting directly on the sensory neuron. Interestingly, the discovery of a far greater potency of calcitonin gene related peptide (CGRP) in producing migraine pain-like behaviors in female mice also involves a peptide with intimate connections to nociceptor biology (Avona et al., 2019).
In conclusion, our data demonstrate that sensory neuronal Prlr contributes to female-selective regulation of hypersensitivity in inflammatory pain models via local and spinal mechanisms. Sex dependency of PRL responsiveness and the generation of hypersensitivity by this mechanism in sensory neurons is likely controlled via translation mechanisms.
We favor the hypothesis that sex-specific regulation of sensory neuronal excitability by PRL is governed via E2-controlled translation regulation of Prlr mRNA, and this in turn explains female-selective mechanisms for regulation of PRL-induced hypersensitivity in non-injured animals and inflammatory pain by sensory neuronal Prlr.
These results add a new layer to our understanding of sex dimorphisms in pain signaling and further substantiate the critical role that translation regulation plays in setting nociceptor excitability in response to a broad variety of important physiological stimuli.
University of Arizona