Objective Vocational rehabilitation measures support youth and young adults with disabilities to obtain vocational training and to enter the labor market. In Germany, a growing number of young people with psychological disabilities in vocational rehabilitation can be observed. The study at hand focuses on this group and examines their (un-)unemployment biographies before vocational rehabilitation, their access to vocational rehabilitation and identifies their individual challenges within the process of vocational rehabilitation. Methods Using a multi-methods approach, we analyze representative administrative data of the German Federal Employment Agency as well as biographical interviews conducted with young rehabilitants. We compare the population of young rehabilitants with psychological disorders to those with other disabilities in terms of vocational rehabilitation and initial labor market entry in order to get a representative picture about their school to work transitions. Since rehabilitants with psychological disabilities tend to be older than the remaining population, analyses are stratified by age groups. In addition, qualitative in-depth interviews provide an additional and deeper understanding of specific employment barriers youth with psychological disorders have to overcome. Furthermore, the individual perspective gives insight on how the crucial transition from school to work is perceived by the population under study. Results The pathway into vocational rehabilitation of youth with psychological disorders is often characterized by obstacles in their transition from school to work. During rehabilitation, it appears essential to provide psychological stabilization along with vocational training. Although their average level of education is higher than those of other rehabilitants, labor market transition after  vocational training challenges many young people with psychological disabilities, leaving many of them with comparatively poor labor market prospects. Conclusions Young persons with psychological disabilities, who come from regular schools or dropped out from regular school or university, seem to find their way to vocational rehabilitation more indirectly. Furthermore, vocational rehabilitation itself is often prolonged for those with psychological disabilities possibly due to a corresponding stabilization process. However, vocational rehabilitation can be a core element within the stabilization process of a psychological disease. Employment is of primary importance to financial security and social well-being in Western societies . There iIn our exploratory study, we focus on (a) and (c) and analyze rehabilitation programs for youth and young adults with psychological disabilities and their transition to the labor market. Vocational rehabilitation aims at improving employment prospects after a phase of incapacity to work and for people with disabilities. Furthermore, in Germany, vocational rehabilitation is intended not only to help people to return to work, but also to help young people in their initial labor market entry.Transition from youth to adulthood is associated with several challenges. During adolescence, young people are expected to graduate from school, complete vocational training or university, and find their first job and, in addition, move into their first own apartment, have partnerships and start a family. For vulnerable groups, e.g., adolescents with disabilities, these challenges are even more difficult, because they also have to manage limitations due to their disability , 9. AdolIn Germany, the Federal Employment Agency (FEA) is responsible for the initial labor market integration of individuals with disabilities. According to Sect. 2(1) of Book IX of the German Social Code,a person has a disability if their physical ability, mental capacity or mental health is likely to fall short of that typical for their age for at least six months and their participation in society is consequently impaired. They are deemed at risk from disability if such impairment is anticipated.Individuals with disabilities or at risk of disability are legally entitled to apply for vocational rehabilitation. In order to support youth with disabilities during their school to work transition, vocational rehabilitation provide participants with career orientation and vocational training.Pathways to vocational rehabilitation are highly institutionalized for students at special schools. About 1 year before graduation, the rehabilitation personnel of the local employment agency (LEA) visit special schools and provide information on vocational rehabilitation opportunities and support for vocational preparation and training.Nevertheless, in Germany over the last decade, rehabilitation requests from youth with psychological disabilities have increased. While youth with learning disabilities remains the largest group among young rehabilitants (about 50%), the share of rehabilitation participants with psychological disorders increased from 15% in 2009 to over 20% in 2014 .The increasing share of youth with psychological disorders is not surprising. Over recent decades, psychological diseases have been increasingly diagnosed, the public has become more aware of psychological disorders, and social acceptance increased . The notMental disorders and psychosocial impairments often occur in youth. The episodic and persistent courses of most psychological diseases particularly challenge those affected in terms of their labor market integration . TherefoIn the study at hand we analyze the specifics of the vocational rehabilitation process experienced by young people with psychological disabilities. We focus on those in training measures and apply a multi-methods approach. An analysis of the FEA’s administrative data allows us to provide a representative picture of individuals with psychological disorders, their entries to vocational rehabilitation and to illness-specific labor market programs. Using representative data, we are able to identify general institutional circumstances and obstacles young men and women with psychological disabilities are confronted with during initial labor market integration. Contrasting their typical situation with those of men and women with other disabilities might indicate whether or not appropriate rehabilitation measures are available or allocated to the target group .Furthermore, we use qualitative in-depth interviews in order to detect potential explanations for differences found in the quantitative analysis and to give initial advice on how future programs must be designed to best suit the particular situation of youth and young adults with psychological disabilities.Explorative interviews conducted with the target group emphasize the individual perspective of these young rehabilitants with psychological disabilities, their reflections on access to vocational rehabilitation and their experiences during the process of vocational rehabilitation.Though, there are some studies particularly examining persons with psychological disabilities , most stOur exploratory inquiry focuses on the following research questions.Based on administrative data, we ask, how the group of young rehabilitants with psychological disorders differs from rehabilitants with other disabilities in terms of their sociodemographic structure and typical rehabilitation process patterns.2.Thus, we further ask why rehabilitation patterns differ for those with psychological disabilities. Based on qualitative interviews, we therefore generate further hypotheses on how young people with psychological disorders perceive the process of vocational rehabilitation, which typical obstacles they have to overcome before entering vocational rehabilitation and during the process.Sociodemographic differences as well as extended periods of transition and rehabilitation duration might suggest illness-specific needs and the unavailability of appropriate rehabilitation programs in accordance with Baron and Salzer .2.Thus, In order to analyze the specific situation of youth with psychological disabilities in training measures for initial labor market integration, we make use of different data sources and follow diverse research approaches. First, we use consolidated administrative data collected in the vocational rehabilitation processes covered by the FEA (German Reha-Process Data Panel (RehaPro)). The data is comprised of all individuals with periods of vocational rehabilitation covered by the FEA between 2007 and 2014   the transition from school to rehabilitation: the period after the end of schooling, the entry towards vocational rehabilitation and the entry towards the first rehabilitation program after being acknowledged as a rehabilitant, (2) the rehabilitation phase itself: the duration of vocational rehabilitation and vocational training  and the labor market entry. In order to account for descriptive differences, we stratify multivariate analyses estimating the two process periods before and during vocational rehabilitation by age groups . Afterwards, results of the qualitative analysis are presented to provide an individual perspective on psychological disorders and educational biographies and to gain a deeper understanding of how different obstacles are perceived by the population studied for the different steps of the process.Table In comparison to youth with other disabilities, those with psychological disabilities show longer periods of unemployment after the end of school. However, stratified analyses by age groups reveal that the result is only valid for those under the age of 20 Table , model 1Generally, the period between the end of school and the start of the rehabilitation process is longest for people with psychological disabilities. Only among those at the age of 25 and older, schooling time has passed considerable longer among persons with a mental disability Table , model 2If persons are acknowledged as a rehabilitant, it generally takes about two months until the first program starts. Persons with psychological disabilities do not differ from persons with musculoskeletal disabilities in this context over different age groups, but they participate in programs a few days later than persons with mental disabilities  and persons with a learning disability  Table , model 3rehabilitation of people with psychological disability is significantly higher than the duration of individuals with learning, mental or musculoskeletal disabilities. This applies for almost all age groups except for persons younger than 17 years of age where persons with a learning disability stay slightly longer in vocational rehabilitation , whereas others are provided with vocational training in special vocational training centers with different degrees of in-company experience. German vocational training generally comprises a theoretical and practical part. Rehabilitants attaining in-company vocational training complete the theoretical part at regular vocational schools; the practical part is completed within the company providing the apprenticeship. This type of training corresponds to the regular form of vocational training within the German dual system.However, most rehabilitants participate in vocational training at rehabilitation-specific and company-external vocational training centers, where theoretical and practical training is provided at the vocational training center (integrative training model) or where the practical part of the vocational training is provided in cooperation with a company (cooperative training model). Integrative training is the most common type of vocational training provided under vocational rehabilitation, particularly among youth with psychological disabilities see Fig. .Generally, the FEA considers the process of vocational rehabilitation as completed if stable employment, defined as employment for more than 6 months within half a year, is obtained. If a rehabilitant does not enter employment, other outcomes or reasons for ending rehabilitation are noted by the rehabilitation personnel—most commonly sickness or a lack of cooperation. In comparison with rehabilitants as a whole, individuals with psychological disabilities have a considerably lower success rate in obtaining employment (16 vs. 25% in the remaining population). Furthermore, they are more likely to end rehabilitation due to medical reasons. As many as 20% of rehabilitants with psychological disabilities exit vocational rehabilitation due to medical issues (compared to 4% in the remaining population) (without illustration).The aim of the qualitative analysis is to generate hypotheses on reasons and explanations for the quantitatively outlined differences between young rehabilitants with psychological disabilities and other young rehabilitants. At the beginning of each interview the interviewer asked the interviewees to describe their access to rehabilitation and their experiences during schooling. The descriptions of the transition phase from school to rehabilitation suggest that most obstacles to vocational education and employment are rooted in their psychological condition. Accordingly, the interviewees reflect on the beginning of their psychological disorder, the c course it has taken. They describe that they struggled at school and dropped out before graduation:“In the eleventh and twelfth grade I wasn’t able to focus on my school stuff, ... and so then I thought I needed help from someone outside and I first went to hospital, but they couldn’t help me so much there, I was there six, eight weeks and then I went back to school and after about six months in twelfth grade, because I was failing [at school] because of my illness, I ended up in hospital” (EEG_L_b)The interviewees associate pressure experienced at school as well as during periods of transition between different schools and/or (pre-)vocational training and unsuccessful attempts to find vocational training or employment, difficult family situations and typical problems during puberty with the occurrence of their psychological disorders. Psychological conditions and challenges in the school to work transition can be interpreted as mutually reinforcing and lead to long periods of inactivity:“... and then I tried to find an apprenticeship [i.e. vocational training] and that didn’t work at all and by the time I left school I already had exam anxiety and such like and that was all made worse by the rejections; and that’s when my social phobia really took hold ... for four years I didn’t really do anything because I didn’t even try and I was also in therapy...” (EEG_M_d)Interviewees also reflect on their previous biographies and medical histories, explaining how their illnesses dragged them down. The description of a 25-year old man with a gambling addiction illustrates the association between the course of psychological disorder and labor market integration process over a long period for those attending mainstream schools on a higher educational level. He summarizes his personal collapse and his several attempts to find vocational training and employment:“And at some time (while I was at school) I started going regularly to the amusement arcade... I was pretty addicted. …And then I felt so bad, I didn’t go to school at all. … At school they knew that I was addicted to the machines, and I’d already repeated a year and then despite that they offered me the chance to do another year again. But then, I thought, it’s not really worth it because my grades are so bad. And that’s why I thought, I’ll get an apprenticeship [i.e. vocational training]. … So, I started this training ... but it wasn’t a good time for me, I wasn’t really part of the team, I had difficulties communicating and I was still in the amusement arcade all the time, my wages all went on the machines ... I got through the trial period, but after four months I quit my training, did a bit of casual work and then I wanted to get my high school diploma [Abitur] at the technical high school [Fachoberschule], so then I went to high school ... but I didn’t want to admit to myself that I was in a really bad state. … I could hardly concentrate. So, of course I didn’t get through the trial period at high school. Then I took a mini-job. … But this mini-job wasn’t enough for me and so I wanted to start another apprenticeship. Then I tried to do the training at the same time as my mini-job. And that didn’t work either. And then things just got worse and worse. At some point, I couldn’t even get myself to work anymore... And there I was - simply at home.” (EEG_L_d)However, when taking school types into account, two different transition patterns occur. While longer periods of illness, unemployment and unstable employment contracts are reported from those attending regular schools, attendees of special schools describe their transition into vocational rehabilitation as comparably institutionalized. They learn about the possibility of vocational rehabilitation from career counselors of the LEA at school. The description of a 20-year old special school graduate illustrates the institutionalized transition process:“I just got this letter from the Local Employment Agency telling me that I was transferring to a one-year pre-vocational program [Berufsvorbereitungsjahr] … I got a year’s training there as a metalworker. And after that, after the pre-vocational program, I got a letter again, this time inviting me to the next preparation program [berufsvorbereitende Bildungsmaßnahme = pre-vocational training].” (EEG_M_g)Students attending special schools seem to be diagnosed with psychological disorders early in their life course. Among attendees of regular schools, psychological disorders often seem to occur later during school attendance and increase the chance of interruptions or dropouts from school, possibly due to a phase of disorientation. Attendees of regular schools seem to learn about vocational rehabilitation from others than professional career counselors, e.g., from medical professionals during hospitalization as a 25-year old woman who dropped out of university reports:“At hospital I was shown various options of what I could do next and one of them was a vocational rehab program and I said that doesn’t sound bad. ... I’m glad that in the hospital they emphasized this opportunity” (EEG_L_a)In order to prepare for vocational training, specific preparation measures that combine medical and vocational rehabilitation are available for those with psychological disabilities. These measures take place in specialized institutions—known as RPK units.A female rehabilitant describes the wide range of therapies offered within a RPK unit. In addition to one-to-one sessions and group sessions, she was also offered practical training such as communication training . She reflects:“At that place there isn’t anything that’s compulsory, they simply offer you these different therapy options and they’re really useful, most of them are really useful if you actually use them. Yeah, there’s plenty on offer.” (EEG_L_a)Besides psychological and medical stabilization and occupational orientation, preparation measures at RPK units allow for a gradual adaption to the schedule of working life. As the following quotation illustrates:“At the beginning I had the problem, I’d not done anything for four years, and simply to get into the rhythm of work ... and so the way I got started was at first just going there in the morning until 12 and then I increased that, getting up to the whole day by the time I started my apprenticeship. And now the apprenticeship’s started it’s not a problem anymore.” “...the pre-vocational program was a great way of building up to a full daily routine and a full working day.” (EEG_M_d)Since periods of illness, hospitalization and disorientation are described to be accompanied by a loss of social contacts and isolation, gradual adaption to interaction with others seem to further contribute to individual stabilization.Furthermore, active participation in a working environment and the experience of success and improvement appear beneficial for recovery. Participation in vocational rehabilitation therefore seems to promote the sense of self-awareness and self-empowerment of young rehabilitants as the following quotation of the same interviewee illustrates:“And in my case, I think things, they can only get better if I, if I do something. And that I manage to achieve something and somehow get back some self-confidence. And, in fact, that’s the only reason, I think, that I’ve got better.” (EEG_M_d)After orientation and stabilization measures, often vocational training measures proceed in order to gain sustainable employment. As described above, these measures can be organized in a company-external or a regular in-company manner. Our sample is only comprised of individuals with company-external training measures.“Completing ‘normal’ company-internal vocational training would possibly not be successful. … I would fail again. … I am not completely fit. … I would like to complete this vocational training [within the vocational training center].” (EEG_L_d)This quotation of a 25-year-old man illustrates a high level of self-reflection on the own abilities and therefore the wish to attend a company-external training measure. Besides possible restrictions caused by the course of the disease, own abilities and personal preferences as well as the educational level or former school dropouts determine the choice for company-external vocational training.Within the phase of vocational training the coping process continues as a 24-year old man describes:“I was afraid of situations, in which I did not know what to expect. … I absolutely tried to settle every possible scenario. … This [behavioral pattern] slowly diminished. … As I am now heading towards job search and job interviews, I do not know [how this will be]; I can barely assess that myself, but at the moment during my vocational training I don’t have any problems, anymore.” (EEG_M_d)In sum, rehabilitants report of the vocational rehabilitation as a phase that provides stabilization and job orientation.In Germany, about one-fifth of all young rehabilitants are diagnosed with a psychological disability. The size of this group has been growing over recent years. Our study is the first to present a broad picture of vocational rehabilitation during the labor market entry phase of youth with psychological disabilities in Germany. The use of administrative data allows us to compare the population studied to young rehabilitants with other disabilities to point at special needs before, during and after the rehabilitation process. The administrative data also includes information on school and employment trajectories before vocational rehabilitation. Thus, we portray a complete picture of their way into vocational rehabilitation. In addition, enhancing the information available in the administrative data with qualitative interview data we are able to provide a deeper understanding of the individual ways into vocational rehabilitation and the motivation, self-assessment and challenges of youth with psychological disabilities during their labor market entry phase.school to work transition phase, the results show that school to work transitions constitute a particularly vulnerable phase in the life course of youth with psychological disabilities. Although in general, school to work transitions become longer [First, looking at the e longer , those wAccordingly, our results show that already at a very young age many rely on social benefit receipt. The qualitative interviews reveal many different obstacles these young men and women have to overcome, such as difficult family situations, serious struggles at school and lack of social as well as financial support. Also, it seems that in contrast to other disabilities, psychological disabilities often get diagnosed during the school to work transition phase. Therefore, further research should emphasize on the social situation of youth with psychological disabilities and test for causality between social circumstances and disability management.Qualitative interviews also suggest that personal development of youth with psychological disabilities is hampered by the impairment and difficulties in finding a career perspective. In addition, the psychological disability aggravates their situation and psychological obstacles hinder finding a career. It takes time to recover or to learn how to cope with psychological issues. Therefore, a deeper perspective towards the relationship between psychological stability and social development during school to work transitions is necessary.In this context, the qualitative interviews further suggest that there are at least two different patterns of transition. On the one hand, we can identify a group of students from special schools with gapless transitions from school to vocational rehabilitation. They represent a group who was affected by psychological disorders already earlier in their life course. This leads to special school attendance, close supervision and institutionalized intervention at the end of schooling, e.g., in early access to vocational rehabilitation. On the other hand, for students coming from mainstream schools or university with longer transition periods, phases of illness and recovery, late entries to vocational rehabilitation and school drop-outs can be observed. They tend to get diagnosed with a psychological disability at a higher age. Further research is necessary whether or not these two groups can also be identified in a representative sample in order to implement targeted support to provide early access to institutional support that can ease school to work transitions.For instance, the qualitative interviews hint at problems in identifying rehabilitation needs possibly explaining the statistically identified longer transition rate from school to vocational rehabilitation. The FEA seems to have trouble in reaching youth with psychological disabilities, especially when they are visiting regular schools. However, it seems important to analyze how graduates and school drop-outs are supported in their transition phase and which special offers as well as “early warning systems” might help to reach out to those with psychological disabilities—at best before psychological disabilities manifest.the rehabilitation phase, the quantitative results show that youth with psychological disabilities remain longer in rehabilitation as well as in vocational education during rehabilitation than those with other disabilities . The qualitative interviews indicate that preparation and stabilization measures are necessary for those with psychological disabilities potentially prolonging the process of rehabilitation. In special treatment measures and company-external vocational rehabilitation, youth with psychological disabilities learn to adopt daily routines and to interact with customers and colleagues.Second, with regard to transition into employment after completing vocational education, young men and women with psychological disabilities show lower transition rates into employment than those with other disabilities. Thus, on the one hand, a lack of appropriate jobs can be assumed. On the other hand, again a particularly vulnerable transition phase has to be overcome which is not necessarily successful.Finally, with regard to the In the overall discussion of our findings, the following limitations must be considered. First, although our quantitative analysis was conducted with a full census of administrative data, administrative records lack detailed information, e.g., on specific occupational restrictions due to the registered disability and on the complexity of health situations. Second, qualitative interviews were only conducted with a small sample of rehabilitants and, thus, analyses do not claim to be representative for the whole population studied. However, the complex realities of individuals with psychological disabilities can only be understood through individual perspectives. When sampling the interviewees, we followed the principle of contrast and chose interviewees with different characteristics with regard to gender, age, educational background and institutional setting . TherefoThe current study shows that among young persons with disabilities, those with psychological disabilities are particularly confronted with obstacles during their labor market transition. In Germany, vocational rehabilitation is meant to assist youth with disabilities in order to obtain vocational education and sustainable employment. Our exploratory approach sheds light on the growing group of young people in vocational rehabilitation with psychological disabilities. However, our results hint at access difficulties to vocational rehabilitation as well as difficulties during and after vocational rehabilitation among youth with psychological disabilities. Thus, recognition of psychological health problems needs to be improved and eligibility for vocational rehabilitation should be granted already in school. In addition, an overall system supporting school drop-outs and youth in early unemployment with psychological health problems could help to improve employment prospects of youth with psychological disabilities.Furthermore, longer durations of rehabilitation and vocational education within rehabilitation might hint at further difficulties of youth with psychological disabilities. In this context, company-external vocational training including preparation and stabilization courses might prolong the process of rehabilitation. The higher share of rehabilitants with psychological disabilities quitting rehabilitation due to health issues further reflects the episodic character of psychological diseases . SpecifiFinally, our results show that future qualitative and quantitative studies on vocational rehabilitation and the subsequent labor market transition process should take persons with psychological disabilities and their specific needs particularly into account. In this context, besides individual also external obstacles to employment, such as attitudes of employers towards people with  disabilities should be taken into account.


We got together for Prof. John B King’s funeral on 26 October 2018 in Thurlestone, near Kingsbridge, Devon. Prof. King had a full life and died of complications following aortic aneurism surgery.Prof. King’s name has been inseparable from that of the Queen Mary University of London for many years. John King qualified at the then London Hospital Medical College in 1967. He completed his orthopaedic training on the London Hospital programme, with time spent in the knee unit at the Royal National Orthopaedic Hospital. Prof. King was a precursor: at a time when British orthopaedic surgery was open only to ideas from the other side of the pond, he understood that the other side of the channel had much to offer, and he spent time in Lyon on a fellowship with Professor Albert Trillat, one of the foremost knee surgeons in the globe.Prof. King was appointed Senior Lecturer in Orthopaedic and Trauma Surgery to the London Hospital Medical College  and Honorary Consultant in Orthopaedic and Trauma Surgery to the London Hospital and held this position from 1977 to 2001.Prof. King’s activity as a trainer in trauma and orthopaedic surgery has brought him in contact with a host of unruly young registrars to whom he communicated the calm feeling of the man in charge and the knowledge that he was always there for them. He has produced a cohort of consultant trauma and orthopaedic surgeons of the highest calibre, who retain the utmost respect for the man who introduced the art and the science of arthroscopy in this part of the world.Prof. King was a man of great vision and a hard taskmaster. When I first reached the UK from Italy, going through a booklet produced by the General Medical Council, I discovered that the only place in the UK where one could learn sports medicine was the London Hospital. In 1981, the then Mr. John King started, with the help of the powers that be of the College, the Diploma Course in Sports Medicine. It soon became ‘the course’; subsequently, the Academic Department of Sports Medicine was founded, and it is now the Centre of Sport and Exercise Medicine at Barts and the London School of Medicine and Dentistry, Queen Mary University of London. Even in the early part of this century, the course remained the only way in the whole of the British Commonwealth to learn in a concerted fashion sports medicine. The diploma course became the Master of Science in Sport and Exercise Medicine, and it has a younger brother in the Intercalated Bachelor of Science in Sport and Exercise Medicine.I met John King in person for the first time at the interview for the position of Lecturer in Sports Medicine and Registrar in Orthopaedics at the Institute of Child Health and the Hospital for Sick Children in London, with some sessions at the London Hospital. I remember that day well: with Mr. King, Prof. Phillip Graham, Prof. Michael Preece, Prof. June Lloyd and Mr. Stephen Rowley sat on the formidable panel. I was at the time a basic surgical trainee, wishing to pursue a career in orthopaedics, strongly biased towards sports medicine. My consultant at the time, Mr. JP Green, in Boston, Lincs, told me that I stood little chance to get the job. I was young, inexperienced and not trained in the UK, and sports medicine was but a little part of trauma and orthopaedics. His reference must have said good things about me, and I was offered the position. I remember that Mr. King came out of the interview room and, given my past as a researcher in molecular biology, with a great smile congratulated me and told me ‘Next time we see, you will tell me about Western blotting’. I made sure that I knew about it, but Mr. King never asked me again.Following that interview, Mr. King supervised my PhD at the Institute and started training me in arthroscopy and sports medicine. I remember that we performed the first arthroscopy of the ankle together, in 1987, and, a few weeks later, we had a look inside the shoulder. The knee was commonplace, and there were times when he undertook 30 operative knee arthroscopies per week.I was examined by Mr. King more than I like to remember: he was my internal examiner for my Master of Surgery thesis, my external examiner for my MD thesis and the examiner for my Diploma in Sports Medicine, both in Edinburgh and at the Apothecaries. He appointed me as a registrar in the North East Thames Orthopaedic Rotation, and all the time, he pushed me.A few weeks after I started my lectureship in sports medicine, he told me ‘Nick, bone always heals. Cartilage never heals. Where we can make a difference is in muscles, ligaments and tendons’. It was a no-brainer, and my interest in soft injuries was born. John made sure that I however undertook my ‘normal’ orthopaedic training, and in this way, I would never be shortsighted.John was never short of advice. When I finished my Registrar training, and I was looking for a Senior Registrar post, he suggested that I applied outside of London. ‘People outside of London do not know you, but you are good. They will shortlist you, you will get great interview experience. But you will not be appointed, so by the time the right job will come in London you will blow them away with the answers you will have learnt from not getting the jobs outside of London’. I took his advice, and, not wanting to risk to not be in London, I applied to the Senior Registrar job as further away as possible, Aberdeen. Within 3 weeks of the application, I had been shortlisted. I spoke about it to Mr. King and to the other great John in my professional life, Mr. John Fixsen. They drilled me well, and 1 week later, I was in the far North East in the UK to be interviewed. I was offered the job. I did not know what to do and phoned Mr. King: ‘You bloody fool, just say yes’. And so another great part of my life came forward, and I moved to Scotland. At the time, in December 1992, there was no Internet, but I came down to London more often than not and kept in touch.John told me that he would have supported my return to London, and he did try to establish a Senior Lectureship at the London with an interest in the upper limb. He came to visit me in Hong Kong, while I was an Associate Professor over there. On that occasion, Mrs. King, Deidre, told me to stop addressing her as ‘Mrs King’, and Mr. King asked me to address him as ‘John’. An honour I have been proud since.Without the work that Prof. King pursued along the years, the UK would not have Sport and Exercise Medicine as a full-blown specialty: the course that he started in 1981 culminated a quarter of a century later with specialty recognition. To Prof. King the thanks from the whole Sports and Exercise Medicine community of the UK, whom he led when he was at the helm of the British Association of Sports Medicine.Prof. King left his position of Senior Lecturer in Orthopaedic and Trauma Surgery in 2001 but has been present in the Centre for Sport and Exercise Medicine ever since; we made sure of that, and we continued to involve him by asking to lecture and examine on the course, to supervise research projects and to exert his clinical acumen with the patients the management of whom we did not have not a clue about!In 2008, Mr. King convinced the powers that be that the Centre for Sport and Exercise Medicine needed a Professor. I successfully applied. When I took up post, in one congress, Mr. King commented publicly that I was now senior to him. This could not be, and in 2011, I made sure that Mr. King was granted an Honorary Professorship for his involvement with the course: a long due position for the man who led the British Association of Sport and Exercise Medicine and was awarded the Sir Roger Bannister Medal for Lifetime Services to Sports and Exercise Medicine.Over the years, I developed more and more respect for the surgeon who trained me not just as a surgeon, but as a person and a doctor. I often cite Prof. King’s maxims and aphorisms and remember his smile and his wit. I sort of envy his style and teach my trainees what remains at the basis of what Prof. King instilled in me ‘Learn principles and concepts, not techniques. Principles stay, and you are great. Techniques change, and, without concepts, you are stuffed’.Prof. King always maintained his family close: the love for his wife Deidre, for his children Polly and Toby and his grandchildren has always been foremost. And he has enlarged his family by embracing all the guys he trained, and at times their children. I know it: my own son, Giuseppe, is John’s godchild!


SLNCR and increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes. ChIP-seq reveals that ligand-free AR is enriched on SLNCR-regulated melanoma genes and that AR genomic occupancy significantly overlaps with EGR1 at consensus EGR1 binding sites. We present a model in which SLNCR recruits AR to EGR1-bound genomic loci and switches EGR1-mediated transcriptional activation to repression of the tumor suppressor p21Waf1/Cip1. Our data implicate the regulatory triad of SLNCR, AR, and EGR1 in promoting oncogenesis and may help explain why men have a higher incidence of and more rapidly progressive melanomas compared with women.Melanoma is the deadliest form of skin cancer, affecting men more frequently and severely than women. Although recent studies suggest that differences in activity of the androgen receptor (AR) underlie the observed sex bias, little is known about AR activity in melanoma. Here we show that AR and EGR1 bind to the long non-coding RNA   Long non-coding RNA function can be understood by defining their interacting proteins. Schmidt et al. demonstrate that the melanoma lncRNA SLNCR binds to AR and complexes with different transcription factors to mediate invasion or proliferation. Thus, lncRNAs regulate distinct transcriptional programs based on specific protein interactions. The worldwide incidence of melanoma has been on the rise for the past 30 years. In the United States, there are ~73,000 new cases diagnosed and ~10,000 deaths annually attributed to melanoma . Of thesHOTAIR  regulates tumorigenesis in many human cancers, including prostate, breast, kidney, lung, bladder, and liver cancer . AR may HOTAIR , or by pHOTAIR . Early sHOTAIR . In direHOTAIR . InteresSLNCR) function through direct interactions with proteins. Using a highly sensitive technique developed for the identification of RNA-associated transcription factors called RATA (RNA-associated transcription factor array), we showed that SLNCR binds to multiple transcription factors, possibly regulating their downstream transcriptional activities  as a candidate SLNCR1-interacting transcription factor  to knock down all SLNCR isoforms  (SLNCR by ~60%–80% in two patient-derived melanoma short-term cultures (MSTCs), WM1976 and WM858, and by 50%–70% in the immortalized malignant melanoma cell line A375  . These sine A375 . Importaine A375 . WM1976 A or RNA . Using RSLNCR expression is significantly correlated with expression of 120 candidate target genes , strongly suggesting that our RNA-seq analysis faithfully identified many SLNCR-regulated genes.Analysis of the full melanoma dataset from The Cancer Genome Atlas (TCGA) revealed that SLNCR differentially expressed genes (DEGs) to isoform-specific SLNCR1 DEGs identified previously  were also dysregulated upon knockdown of SLNCR1 (SLNCR and SLNCR1 DEGs (35 of 41) displayed concordant dysregulation  and integrin subunit beta-1 (ITGB1), regulate cell matrix adhesion , whereas another two of these genes, transmembrane protein 45A (TMEM45A) and Ras-related protein RAB31, have been implicated in cancer invasion and cell adhesion  SLNCR knockdown identifies significant enrichment of genes involved in multiple cancer-relevant processes, including cell adhesion and/or motility, apoptosis, differentiation, response to stress, and proliferation , likely because of a lower endogenous levels of SLNCR, which limits the fold depletion and range of knockdown-related phenotypes  or are the reverse complement to the SLNCR sequence required for AR binding, which generates double-stranded RNA incapable of binding AR (antisense 1 or 2). The antisense oligonucleotides are specifically designed to bind to SLNCR without eliciting RNase H-mediated degradation of SLNCR. Gymnotic delivery  of either AR-or SLNCR-binding 2′-deoxy-2′-fluoro-D-arabinonucleic acid (2′-FANA)-modified oligonucleotides significantly decreased melanoma proliferation without decreasing SLNCR expression . Decreased cell proliferation upon inhibition of the SLNCR-AR interaction, despite increased SLNCR expression, further indicates that SLNCR and AR cooperatively regulate melanoma proliferation.To test whether pression  and S3D.SLNCR1 regulates AR occupancy at at least one genomic region  in hormone-deprived A375 melanoma cells  to identify global genomic loci bound by ligand-free AR . Performing AR ChIP-seq from MSTCs is technically challenging because of low AR expression; thus, we performed AR ChIP-seq from higher AR-expressing A375 melanoma cells. Because c region , cells wma cells . The majSLNCR1 regulates AR chromatin occupancy. SLNCR1 overexpression (1) increased the number of AR active regions  without increasing expression or altering localization of AR  incon of AR , (3) incon of AR , and (4)on of AR . Collectd SLNCR1 , we consSLNCR-regulated genes, we searched for genes that are both AR-bound , as determined by AR ChIP-seq  were bound by AR, but 43.2% of SLNCR-regulated genes (96 of 222) were bound by AR , and 43.8% (53 of 121) of genes dysregulated by SLNCR1 knockdown were bound by AR , further supporting that AR binding is enriched on SLNCR- and SLNCR1-regulated genes , JUN oncogene , STAT3 transcription factor , interleukin-24 , and melanoma cell adhesion molecule . qRT-PCR of several AR-bound, SLNCR-regulated genes  before and after siRNA-mediated knockdown of either SLNCR or AR confirms that SLNCR and AR regulate expression of these target genes in both WM1976 and A375 cells , suggesting that SLNCR and AR may regulate the expression of certain target genes in an opposing manner. Integrative analysis of SLNCR RNA-seq and AR ChIP-seq datasets reveals that AR binding is enriched on SLNCR-regulated genes and suggest that AR and SLNCR similarly regulate the expression of many of these target genes both in vitro and in vivo.Many of the AR-bound, 75 cells . ContrarSLNCR-regulated genes (148 of 222), 66 of which are also bound by AR based on our ChIP-seq analysis , further supporting our hypothesis that SLNCR and AR cooperatively regulate the expression of many of these target genes in vivo.Analysis of TCGA expression data reveals that AR is significantly correlated with expression of over half of analysis . CorrectSLNCR-mediated regulation of one representative gene. We focused on the SLNCR-mediated regulation of CDKN1A, the gene encoding the tumor-suppressive cyclin-dependent kinase (CDK) inhibitor 1A (p21Cip1/Waf1), for several reasons: (1) p21 is an important regulator of cell cycle progression and anti-proliferative pathways, inducing G1 or G2 cell cycle arrest  it e arrest ; and (3)e arrest . Additioe arrest . In agreer cells . AR knocer cells . FurtherTP53) nor protein are significantly altered upon knockdown of AR or SLNCR in WM1976 or A375 (wild-type p53 [p53WT]) cells  primary malignant melanoma cell line SK-MEL-28  cells -S5C. AddSLNCR depletion mimics p21-induced melanoma phenotypes. SLNCR knockdown in WM858 (p53MUT) andWM1976 (p53WT) cells led to an increased percentage of cells in G2/M and a decreased percentage of cells in G1/G0 (SLNCR depletion phenocopies p21 induction of G2/M melanoma cell cycle arrest.We next tested whether in G1/G0 . These dSLNCR (SLNCR knockdown reduced DNA binding of two transcription factors bound to and regulated by SLNCR1 (AR and Brn3a) by 60%, as measured by transcription factor activation array. Decreased DNA binding by AR occurred independently of altered AR expression or localization , E2F-1 (30%), ATF2 (70%), and the ATF2-containing activator protein 1 (API) transcription factor heterodimer (60%)  .SLNCR knockdown decreases DNA binding of known p21 targets, including the estrogen receptor (ER) and C/EBP (both ~40% activity) (SLNCR-interacting protein (SLNCR knockdown downregulates E2F-1 DNA binding by 30%. These data suggest that SLNCR directly (through protein-RNA interactions) and indirectly (through p21-mediated regulation) regulates the activity of multiple transcription factors. Collectively, SLNCR knockdown phenocopies p21-mediated cell cycle arrest and transcription factor regulation, suggesting that SLNCR knockdown induces biologically relevant upregulation of p21.ctivity) , and incctivity) . E2F-1 i protein  that is  protein . SLNCR kSLNCR knockdown dysregulated the activity of multiple transcription factors  and TOMTOM analysis  in RNAs immunoprecipitating with EGR1 . Recombinant EGR1 protein corresponding to amino acids 282–433 altered the mobility of full-length, in vitro-transcribed and biotinylated SLNCR1 in a protein concentration-dependent manner . Collectively, these data further support the conclusion that endogenous SLNCR and EGR1 directly interact in vitro and at endogenous levels in melanoma cells.To distinguish between direct interaction of t manner . InteresSLNCR1 binds to EGR1, and the EGR1 motif is enriched in AR-bound, SLNCR-regulated genes, we hypothesized that AR binds to a subset of SLNCR-regulated genes in cooperation with EGR1. To globally identify EGR1 binding sites in A375 cells, we performed EGR1 ChIP-seq. EGR1 binds a total of 8,373 active regions  and 19.3%  of all genes, respectively, AR bound to 58.8% of EGR1-bound genes  and 46% of SLNCR-regulated, AR-bound genes , whereas significant correlation is maintained for 71 of these genes after correcting for multiple hypothesis testing . Here we comprehensively interrogated the role of AR in melanoma gene regulation, identifying many AR-regulated tumor suppressors and oncogenes. This work suggests that SLNCR have been implicated previously in melanoma invasion, this work defines a role of both in the regulation of melanoma proliferation as well, suggesting that AR and SLNCR may be critical regulators of the RGP-to-VGP transition.Melanoma is a complex and heterogenous disease associated with phenotypically and transcriptionally distinct growth phases. During the radial growth phase (RGP), melanomas proliferate rapidly but are unable to undergo metastasis. Upon transition to the vertical growth phase (VGP), the newly formed tumor begins to grow vertically into the dermis and acquires the ability to metastasize . MoreoveSLNCR function in human melanomas would decrease tumor growth and metastasis. Mouse xenografts are frequently used in preclinical development to test disease mechanisms and model therapy in vivo. We are planning experiments to test SLNCR function in a xenograft model of melanoma. In support of the observations described here, knocking down SLNCR decreases tumor growth and metastasis in mouse xenograft models of lung cancer and hepatocellular carcinoma  as well as modulate preexisting regulatory networks (as for Egr1).Collectively, our data are consistent with a model in which n of p21 . Under nt cancer ,2012. Dupromoter . CollectSLNCR, AR, EGR1, p53, and gender with p21 expression within the TCGA melanoma dataset . Using hierarchical multiple regression, we identified a model containing a significant three-way interaction between EGR1 mRNA, AR, and p53 expression associated with p21 expression ] = 0.12 ; p = 0.008; SLNCR  and a significant two-way interaction between SLNCR and EGR1 , p = 0.024), suggesting an inverse association between SLNCR and p21 expression dependent on the levels of EGR1 and AR. This is consistent with our proposed mechanism where EGR1 is required for SLNCR-mediated repression of p21. Considering our data indicating that (1) SLNCR binds EGR1 and AR (SLNCR or AR knockdown increased p21 expression (SLNCR and AR-mediated p21 regulation (SLNCR-mediated repression of p21 requires AR and EGR1.To further explore the relationship between p21 and known regulatory proteins and RNAs (identified here and elsewhere), we investigated possible associations between 1 and AR , (2) SLNpression , (3) AR gulation  and 6E, SLNCR, not through direct interaction with DNA. AR ChIP-seq peaks at SLNCR-regulated genes display, on average, a lower peak intensity than known AR-targets, consistent with proximal recruitment (as in our model) rather than direct DNA binding. Although current models suggest that trans-recruitment of lncRNA-associated chromatin modifiers occurs through triplex helix formation between the lncRNA and target DNA or through direct base-pairing between the lncRNA and proximally expressed RNAs  sequences enriched among SLNCR-regulated, AR-bound genes do not show significant similarity or complementarity to SLNCR; (2) the CDKN1A locus does not express proximal RNAs in the cells studied here (data not shown); and (3) knockdown of EGR1 does not phenocopy SLNCR knockdown , which would be expected if SLNCR-mediated the EGR1 association to the CDKN1A promoter. Rather, our data are most consistent with a model in which the lncRNA interacts with a protein bound independently to the target loci .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Carl Novina  are from the Wistar Institute collection, A375  and SK-MEL-28  cells were purchased from ATCC. The A375 and WM858 cell lines were authenticated via short tandem repeat profiling at the American Tissue Culture Repository on May 19, 2016. WM1976 and SK-MEL-28 cells were not subject to additional authentication. Unless otherwise indicated, cells were grown in DMEM (Invitrogen) without glutamine supplemented with 10% fetal bovine serum (FBS). Hormone-deprived cells were cultured in phenol-red free DMEM without glutamine (Invitrogen) with 5% charcoal-stripped FBS (Sigma-Aldrich).WM1976 (p53 wild-type) and WM858 , cells were seeded at 0.3 × 104 cells/well in a 96-well plate. Cells were treated with FANA oligos, flutamide, or transfected with the indicated siRNAs 24 hours (hr) post seeding, and proliferation was measured using a 1:10 dilution of WST-1 proliferation reagent (Roche) at the indicated time points. Cells were incubated for one hour at 37°C, and absorbance at 450 nm was measured. For cell cycle analyses, cells were harvested and washed 72 hours post-transfection, fixed in cold 70% ethanol for 2 hours, and incubated in LifeTech PI/RNaseA solution for 30 minutes at 37 degrees. For analysis of apoptosis, cells were seeded at 30 × 104 cells/well in 6-well plate, harvested 72 hours post-transfection, and stained using Biolegends Pacific Blue™ Annexin V Apoptosis Kit with 7-AAD. Cells were analyzed on a Fortessa X-20 and populations were identified and quantified using FlowJo software. For luciferase assays, A375 cells were seeded in 96-well plates at 0.75 × 104 cells per well, transfected 24 hours later with the indicated siRNAs. Fifty micrograms of either wild-type or mutated CDKN1A reporter plasmid and 50 μg of a pCMV-renilla luciferase control vector were transfected Lipofectamine® 2000 (Life Technologies) 24 hours post-transfection of siRNAs. Luciferase activity was measured another 24 hours later using Promega Dual-Glo® Luciferase Assay system. The CDKN1A reporter plasmid was generated by replacing the MMTV promoter in pGL4.36 vector (Promega) using Gibson Cloning (All siRNAs were transfected using RNAiMAX (Thermo Fisher).  Cloning . SequencSLNCR was calculated as the fold enrichment in the IgG or EGR1 IP compared to input control after normalization to the indicated mRNA transcript .AR and EGR1 RIP assays were performed as previously described with minor modifications . For EGRin silico using Active Motif software to a length of 150-200 bp. Fragment density was determined based on the number of reads corresponding to 32-nucleotide genomic bins. Peak calling, to identify intervals with local enrichment in reads, was performed using MACS , supplemented with 5% charcoal-stripped FBS, and transfected with the indicated plasmid 24 hour post-seeding. For EGR1 ChIP-seq, A375 cells were cultured in DMEM without glutamine (Invitrogen), supplemented with 10% FBS, and grown to ~80% confluency. Cells were crosslinked in 1% formaldehyde for 15 min 48 hour post-transfection, and the reaction was quenched by addition of 0.125 M glycine. ChIP-seq was performed by Active Motif using Santa Cruz AR (H-280), or Cell Signaling EGR1 (44D5). After chromatin isolation and fractionation, 75-nt reads were generated by Illumina sequencing (using NextSeq 500) and were mapped to human reference genome (hg19) using the BWA algorithm with default settings. The 3′ ends of aligned reads were extended ing MACS . MACS deSLNCR (1) or si-SLNCR (2) siRNAs, in duplicate, using Trizol. Sequencing cDNA libraries were prepared from 1ug of total RNA using the Illumina TruSeq RNA sample preparation kit (v2). Libraries were pooled and sequenced on the Illumina HiSeq 2500 platform. Normalized read counts (FPKM) were generated in Cufflinks v2.1.1 (http://cole-trapnell-lab.github.io/cufflinks/) by mapping onto the hg19 build of the human transcriptome (https://support.illumina.com/sequencing/sequencing_software/igenome.html). Raw FASTQ sequence was mapped using Bowtie (http://cufflinks.cbcb.umd.edu/), comparing duplicate scramble controls against duplicate conditions of both SLNCR-specific knockdowns. Values represented in the heatmaps were generated by CuffDiff comparison of duplicate scramble controls versus duplicates of only one siRNA duplicate. GeneOntology Enrichment Analysis was performed in MetaCore (Thompson Reuters), against a control background set of genes expressed in skin cells.Total RNA was isolated from WM1976 transfected with either scramble, si-g Bowtie , and difSLNCR-target genes were accessed using cBioPortal (https://www.graphpad.com/). Bonferroni correction for multiple hypothesis testing was performed by defining the significance threshold as the critical p = 0.05 divided by the total number of comparisons. BAM files from RNA-seq and ChIP-seq were visualized using the Integrated Genome Viewer (https://www.broadinstitute.org/igv/)  .SLNCR (1) siRNA, and were harvested and fractionated using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit, according to the manufacturer’s instructions, 3 days after transfection. Ten micrograms of nuclear lysate was used directly as input into the Signosis TF Activation Profiling Plate Array I.WM1976 cells were seeded in 6-well tissue culture treated dishes, transfected 24 hours later with either scramble or si-Unless otherwise indicated, lysate was prepared using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific), according to manufacturer’s instructions. Samples were separated on BioRad Any kD Mini-PROTEAN® TGX Precast Protein Gels and transferred to LF-PVDF using the mixed MW protocol on the BioRad Transblot Turbo. The following antibodies were used: Santa Cruz p53 (DO-1) sc-126 at 1:200, AR (M-20) sc-816 at 1:200, Cell Signaling P21 Waf1/Cip1 (12D1) at 1:1000, Cell Signaling EGR1 (44D5) at 1:1000, Cell Signaling S6 Ribosomal Protein (5G10) at 1:1000, and Cell Signaling GAPDH (14C10) at 1:5000.2, 1 mM DTT), with 2 μg of yeast tRNA, the indicated amount of recombinant EGR1 corresponding to amino acids 282–433 , 0.5 nM final concentration of the biotinylated SLNCR1, and 10 μM of unlabeled SLNCR1 where indicated. Reactions were incubated at room temperature for 20 minutes, 5 μl of loading dye was added, and 20 μl was electrophoresed on Bio-Rad’s 5% Mini-PROTEAN® TBE Gel, 10 well, 30 μl. RNA and protein/RNA complexes were transferred to GE Healthcare Amersham Hybond –N+ Membrane in 0.5x TBE at 400 mA for 30 minutes in 0.5X TBE on Bio-Rad’s Trans Blot Turbo Transfer System. Detection was performed according to LightShift REMSA kit, using Bio-Rad’s ChemiDoc XRS+ System.REMSAs were performed using Thermo Fisher Scientific LightShift Chemiluminescent RNA EMSA (REMSA) Kit, according to manufacturer’s instructions. Briefly, 20 μl binding reactions were assembled in low-adhesion tubes in 1X binding buffer  was downloaded from the NCI Genomic Data Commons (GDC). The RNA-Seq data was in the format of BAM files representing an alignment, using the STAR aligner, of raw reads to hg38. Human gene models were downloaded from RefSeq on November 3rd, 2017, and the SLNCR ranges were defined from the range of LINC00673. For each patient, the total number of reads aligning the SLNCR genomic region was obtained by parsing the output of samtools flagstat and SLNCR-specific counts were normalized by the total number of reads aligning to hg38. The expression values for non-SLNCR genes were obtained by downloading a results table from Xenabrowser (https://toil.xenahubs.net/download/tcga_rsem_isoform_tpm.gz). TPM expression values were computed from a reanalysis of the TCGA dataset under the TOIL framework using RSEM. Hierarchical multiple regression analysis was performed using R v3.2.2, where the model estimates and p values, based on the t-statistic, were calculated using the ‘lm’ function. SLNCR and EGR1 mRNA expression values were log2 transformed to account for non-normal distributions. Expression of AR, p53 and p21 protein was accessed from Level 4 cross-batch normalized data in The Cancer Proteome Atlas (https://www.broadinstitute.org/igv/) , against a control background set of genes expressed in skin cells. Gene expression of AR-and ioPortal . SLNCR Rme Atlas . All conme Atlas . Patientme Atlas . Bonferrrg/igv/) .https://www.graphpad.com/). In proliferation assays error bars represent the mean ± SD of 3 technical replicates. Significance was calculated using the two-way analysis of variance (ANOVA), with the Dunnett test for multiple comparison testing. For binding enrichments, statistical significance was calculated using a either a Binomial test (two-sided) by comparing the observed versus expected probabilities under independence or using a Fisher Exact test of the co-bound targets, using GraphPad Prism software. In cell cycle assays, cell populations were analyzed using FlowJo software, and significance was calculated using GraphPad Prism software. Bars represent the average percent of total cells in the indicated stage of the cell cycle, and error bars represent SD from 3 independent replicates. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Protein levels were quantified using ImageJ, and are presented as a fold change normalized to GAPDH levels. Bars represent mean ± SD from 3 independent biological replicates. Significance for RNA and protein quantification were calculated using the Student’s t test. Significance was calculated using a two-tailed Student’s t test. Transcription Factor Activation Array is represented by relative luminescence mean ± SD from 2 independent biological replicates.t tests, ANOVAs, and correlations were calculated using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA (The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are 12345


Accumulating evidence indicates alteration of the β-adrenoceptor (AR), such as desensitization and subtype switching of its coupling G protein, plays a role in the protection against catecholamine toxicity in heart failure. However, in human takotsubo syndrome (TTS), which is associated with a surge of circulating catecholamine in the acute phase, there is no histologic evidence of β-AR alteration. The purpose of this study was to investigate the involvement of alteration of β-AR signaling in the mechanism of TTS development. Left ventricular (LV) biopsied samples from 26 patients with TTS, 19 with normal LV function, and 26 with dilated cardiomyopathy (DCM) were studied. G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, which initiate the alteration of β-AR signaling, were more abundantly expressed in the myocardium in acute-phase TTS than in those of DCM and normal control as indicated by immunohistochemistry. The percentage of cardiomyocytes that showed positive membrane staining for GRK2 and β-arrestin2 was also significantly higher in acute-phase TTS. Sequential biopsies in the recovery-phase for two patients with TTS revealed that membrane expression of GRK2 and β-arrestin2 faded over time. This study provided the first histologic evidence of the involvement of alteration of β-ARs in the development of TTS. The LV dysfunction is generally reversible with favorable outcome6. Typically, postmenopausal women are susceptible to TTS, which is frequently triggered by intense emotional or a physical stress9. Several mechanisms of TTS have been proposed to date, including multivessel spasm, acute microvascular dysfunction, reactive oxygen species (ROS) production, and catecholamine cardiotoxicity12. In particular, cardiac sympathetic nervous system (SNS) activation has been implicated as an important trigger for developing TTS in studies that showed a significant elevation in plasma catecholamine in patients with acute-phase TTS as compared to those with acute myocardial infarction1, and the elevated catecholamine concentration normalized within a few days15. However, direct evidence of these mechanisms from human tissues was not obtained, nor has the concept been established yet.Takotsubo syndrome (TTS) is characterized by transient hypokinesis of the left ventricular (LV) apex and hyperkinesis of the LV base without coronary artery abnormality18. Over the past two decades, accumulating evidence has shown that the β-AR signaling system itself has several mechanisms of alteration for the protection from catecholamine toxicities, such as receptor desensitization and subtype switching of its coupling G protein from stimulatory Gαs to inhibitory Gαi, which protects from overactivation of PKA, resulting in reduced pumping function21. These mechanisms are initiated by the overexpression of G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, and their subsequent translocation to the cell membrane22.In physiological condition, the SNS regulates both inotropic and chronotropic cardiac functions directly through noradrenaline released from its nerve endings and indirectly through adrenaline released from the adrenal gland via the β-adrenoceptor (AR) in response to body demands. However, in pathological conditions such as heart failure, plasma catecholamine levels are elevated to supraphysiological levels and the subsequent overactivation of β-AR/protein kinase A (PKA) signaling results in various degrees of cardiomyocyte necrosis and apoptosis, termed “catecholamine toxicity”We hypothesized that alteration of β-AR signaling is involved in the mechanism of TTS development. To confirm this hypothesis, we examined the expression profiles of GRK2 and β-arrestin2 in human biopsied samples obtained from the postero-apical portion of the LV, where wall motion was markedly reduced in the acute phase.Baseline clinical characteristics of the three patient groups, including cardiovascular risk factors and medications on admission, are presented in Table Cardiac parameters, obtained by left ventriculography, are presented in Table Laboratory data on admission of the three groups are presented in Table First, we assessed the LV biopsy specimens by hematoxylin-eosin (HE) and Masson’s trichrome (MT) stainings. The degree of interstitial fibrosis in the TTS group was significantly higher than that in the NC group, but similar to that in the DCM group %; TTS, 12.1 (7.8–21.1)%; DCM, 11.3 (4.0–16.0)%, P = 0.005) , whereas in the NC group, fluorescence was largely confined to the cytoplasm Fig. . The GRKImmunostaining of phosphorylated cyclic-AMP response element binding protein (pCREB) was performed to evaluate β-AR/PKA signaling Fig. . The frath day and the other (Case 2) on the 20th day after admission, when LV wall motion and cardiac parameters had recovered to the normal range. In the recovery phase, the GRK2- and β-arrestin2-positive areas in the myocardium were only slightly reduced as compared to the acute phase, from 43% to 38% and from 45% to 38%, respectively, in Case 1, and from 30% to 27% and 44% to 38%, respectively, in Case 2. However, the fraction of cardiomyocytes positively stained for GRK2 and β-arrestin2 on the cell membrane was markedly reduced in the recovery phase from 15% to below the detection limit and from 11% to 1%, respectively, in Case 1, and from 34% to 7% and from 13% to 3%, respectively, in Case 2. Furthermore, the fractions of 8-OHdG-positive nuclei were substantially reduced in the recovery phase; from 27% to 10% in Case 1 and from 23% to 9% in Case 2.Immunohistochemical stainings were also carried out for two patients with TTS in the recovery phase as shown in Fig. 23, this had not been verified in human heart tissue to date. Using human LV biopsy samples from patients with TTS at the acute phase, the present immunohistochemical study demonstrated, to our knowledge for the first time, that GRK2 and β-arrestin2 are overexpressed in TTS cardiomyocytes and distributed throughout the cytoplasm, along with partial translocation to the cell membrane, which are initial steps of β-AR desensitization and subtype switching of the coupling G protein from Gαs to Gαi, leading to reduced LV contraction.While earlier reports assumed an association between SNS activation and TTS19. In accordance with these studies, the present study revealed higher expression of GRK2 and β-arrestin2 in the LV myocardium in patients with DCM. This alteration of β-AR signaling, as indicated by GRK2 and β-arrestin2 overexpression in the LV myocardium, was also observed in acute-phase TTS. Notably, we observed not only higher GRK2 and β-arrestin2 expression in the cytoplasm, but the fraction of cardiomyocytes with membrane-positive staining for both molecules was higher in TTS than in DCM. These observations support the concept that β-ARs on the cell membrane are modified by GRK2 and β-arrestin2, which might play a key role in the development of LV dysfunction in TTS, which presumably prevents catecholamine-induced cardiac tissue damage via a trade-off between cell survival and reduced wall motion. Actually, signals of pCREB, which is phosphorylated by PKA and linked to cardiomyocyte contraction, were not increased in TTS, suggesting β-AR/PKA signaling pathway is not activated in TTS, while those were increased in DCM. Although the PKA signaling has been reported in experimental models of TTS, some reported it as activated as Borchert et al. analyzed in an induced pluripotent stem cells-derived cardiomyocyte obtained from TTS patient23, others reported it as deactivated as Cao et al. studied in an ovariectomized rat model of TTS24, which is still controversial.Alteration of β-AR signaling has been well studied in chronic heart failureIn two patients, we performed a second LV endomyocardial biopsy in the recovery phase, when LV function had been recovered. Samples were obtained from the LV wall, in which wall motion had been severely reduced in the acute phase. In contrast to the acute phase, in the recovery phase, the fraction of cardiomyocytes with GRK2 or β-arrestin2 signal in the membrane was substantially decreased, while the signals in the cytoplasm were only marginally diminished. This serial change in expression profiles of GRK2 and β-arrestin2, especially on the cell membrane, was roughly associated with the decline and recovery of LV wall motion, which indicates the importance of membrane translocation of these molecules in the phenotype of TTS.5. In addition to the transient upregulation of GRK2 and β-arrestin2, their transient translocation to the cell membrane would be characteristic of TTS, and presumably reflects the strength of catecholamine stimulation immediately before biopsy and results in the transient, not persistent, hypokinesis of TTS hearts.When comparing the extent of upregulation of GRK2 and β-arrestin2 expression between TTS and DCM, both molecules were higher expressed in the cytoplasm and cell membrane in acute-phase TTS than in DCM, which is apparently associated with the severer LV hypokinesis in TTS than in DCM. Although the underlying mechanism was not evaluated in the present study, this might reflect a higher and more rapid elevation of the catecholamine levels, termed “catecholamine surge”, in TTS27 and is another factor in transient hypokinesis of the LV in TTS25. Therefore, we investigated the extent of ROS production by immunohistochemistry of 8-OHdG. Acute-phase TTS hearts showed significantly more 8-OHdG-positive cardiomyocyte nuclei than control and DCM hearts. In the recovery phase, the fraction of 8-OHdG-positive nuclei in TTS hearts was reduced to the level in control hearts. Although ROS are produced not solely in response to β-AR signaling, the expression pattern of 8-OHdG was similar to those of GRK2 and β-arrestin2 on the cell membrane, indicating that ROS might also be involved in altering β-AR signaling in TTS.ROS production reportedly is a response to β-AR stimulationBased on our findings, we propose a model on how the studied factors are related in acute-phase TTS in Fig. et al. proposed an interesting hypothesis that hyperkinesis in the LV base with apical hypokinesis may result from the higher tissue density of β-ARs in the apex than in the base20. Namely, the physiological range of catecholamine positively affects LV wall motion through β-ARs, while its supraphysiological elevation could prompt β-ARs desensitization, resulting in LV hypokinesis, especially in the apical portion. Moreover, the β2-AR positive inotropic effect is intercepted by switching its coupling G protein from Gαs to Gαi, which occurs in the presence of a supraphysiological catecholamine concentration, especially, adrenaline, because of the higher affinity of β2-AR to Gαi under elevated adrenaline concentration28. β2-AR-Gαi coupling also activates the phosphoinositide 3 kinase-protein kinase B (Akt) pathway, which exerts an anti-apoptotic effect30. Akt pathway activation in the acute-phase of TTS myocardium has been reported29. In this study, we preliminarily observed the down-regulation of β1-AR expression both in TTS and DCM, while the down-regulation of β2-AR was observed only in DCM. The expression of β2-AR in TTS was as normal level as in normal controls (data not shown). Thus, in the apical LV samples analyzed in this study, β2-AR expression may be relatively up-regulated compared with β1-AR expression, which partially supports the Lyon’s hypothesis of β-ARs distribution and the development of TTS20. Further studies are necessary to elucidate the precise expression profile of β1- and β2-ARs in human TTS patients.The present findings, of course, cannot explain the hyperkinesis of the LV base, which is another characteristic of TTS. Recently, Lyon 3-AR may mediate a countervailing influence on β1 and β2-ARs, resulting in function as “physiological brake” to reduce the effect of sympathetic stimulation32. Given that β3-AR is not downregulated in heart failure34, it remains to be solved that β3-AR is involved in the pathogenesis of TTS.Several studies have reported that the β35. Secondly, only 2 samples were evaluated in the recovery phase. The sample size was too small to lead to any conclusion or perform a statistical calculation, thus larger number of samples is needed for the better understanding of the mechanisms of developing TTS. Thirdly, several papers reported the correlation between GRK2 or β-arrestin2 expression and aging40. In our study, although aging did not affect the expressions of GRK2 or β-arrestin2 in TTS (Data not shown), there might be some influence of aging on the result of increased expression of those molecules in TTS because the age was significantly older in the TTS group. Further investigation is needed to elucidate the relation between aging and the development of TTS. Fourthly, we conducted only immunohistochemistry for protein expression analysis. Generally, it is better to combine western blotting to confirm the result of immunohistochemistry because this technique depends on the specificity and sensitivity of the antibodies used, and is semi-quantitative. In this human study, we could not obtain large samples enough to extract protein for western blotting. To partly compensate for the limited methodology in this study, western blot analysis using autopsied human sample was performed, which showed a single band for each target molecule as shown in Supplementary Fig. There are some additional limitations to this study. Firstly, the sample size was small, and all samples were obtained from a single hospital. However, with respect to the histologic study of human biopsy samples, this study is larger than previous studiesIn conclusion, we provided — to our knowledge for the first time — histologic evidence of the alteration of β-ARs via GRK2 and β-arrestin2 overexpression and their partial translocation to the cell membrane in the acute phase of TTS using biopsied human LV specimens. Transient catecholamine cardiotoxicities and subsequent β-AR’s cardioprotective alteration could be involved in the mechanism of TTS development.41. Among these 69 patients, 26 underwent LV endomyocardial biopsy in the acute phase to exclude myocarditis or other cardiomyopathies, and all of them were enrolled in this study . The endocardium and vascular structure were excluded from the total myocardium area.For immunohistochemistry, anti-GRK2 , anti-β-arrestin2 , WGA-Alexa Fluor 488 , anti-pCREB (Ser133)  and anti-8-OHdG  antibodies were used. Detailed methodology is available in the Data Supplement.GRK2- and β-arrestin2-positive areas in the myocardium were assessed as percentages of total myocardium under the BZ-X710 system. For this quantification, the endocardium, vascular structure, and interstitial tissue were excluded from the myocardium area, and patchy staining area — mainly owing to lipofuscin — was excluded from the positively stained area. To assess GRK2- or β-arrestin2-positive signal on the cell membrane, we counted the number of cardiomyocytes in which GRK2 or β-arrestin2 signal colocalized with WGA, a cell membrane marker, and calculated the proportion of positively stained cardiomyocytes as a percentage of the total number of cardiomyocytes.pCREB-positive and 8-OHdG-positive nuclei in cardiomyocytes were evaluated as a percentage of the total number of cardiomyocyte nuclei, using the BZ-X710 system. These histologic evaluations were carried out by three independent expert cardiologists who were blinded to all clinical data.th–75th percentile) as appropriate, and categorical variables are expressed as percentages. Statistical significance of continuous variables between three groups was assessed with one-way analysis of variance or the Kruskal-Wallis test, followed by the Tukey honest significant difference test or the Mann-Whitney test, as appropriate. Statistical significance of categorical variables was assessed by chi-square analysis. P-values of less than 0.05 were considered statistically significant. JMP software for Windows version 12  was used to analyze data.Continuous variables are expressed as means and standard deviations or medians (25Supplementary Appendix


Aberrant activation of the androgen receptor (AR) may play a critical role in castration resistant prostate cancer. After ligand binding, AR is recruited to the androgen responsive element (ARE) sequences on the DNA where AR interaction with coactivators and corepressors modulates transcription. We demonstrated that phosphorylation of AR at Tyr-267 by Ack1/TNK2 tyrosine kinase results in nuclear translocation, DNA binding, and androgen-dependent gene transcription in a low androgen environment. In order to dissect downstream mechanisms, we searched for proteins whose interaction with AR was regulated by Ack1. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. Interaction between AR and SLIRP was disrupted by Ack1 kinase activity as well as androgen or heregulin treatment. The noncoding RNA, SRA, was required for AR-SLIRP interaction. SLIRP was bound to ARE’s of AR target genes in the absence of androgen. Treatment with androgen or heregulin led to dissociation of SLIRP from the ARE. Whole transcriptome analysis of SLIRP knockdown in androgen responsive LNCaP cells showed that SLIRP affects a significant subset of androgen-regulated genes. Our data suggest that Ack1 kinase and androgen regulate interaction between AR and SLIRP and that SLIRP functions as a coregulator of AR with properties of a corepressor in a context-dependent manner. They include AR gene overexpression/amplification, AR point mutations, AR splice variants and intratumoral androgen biosynthesis. Recently approved drugs such as abiraterone, enzalutamide, and apalutamide, potently inhibit AR signaling in CRPC tumor cells and led to improved outcome of CRPC patients3. However, CRPC inevitably develops resistance to these agents and therefore, metastatic prostate cancer continues to be a terminal disease.Prostate cancer remains the second leading cause of cancer death in men in the U.S. due to the development of resistance to androgen deprivation therapy. Prostate cancer cells remain dependent on the androgen receptor (AR) even in the castration resistant stage (with the rare exception of neuroendocrine tumor). The serum levels of the prostate specific antigen (PSA) protein increase in untreated castration resistant prostate cancer (CRPC) patients as reactivation of AR activity in prostate cancer cells drives the transcription of AR target genes such as PSA. Various mechanisms underlying AR reactivation in CRPC have been demonstrated4. Binding of ligands such as dihydrotestosterone (DHT) to AR causes nuclear translocation and binding to the androgen responsive elements (ARE) of the target genes and assembly of the active transcription complex that contains general transcription factors and AR coregulators5. These coactivators and corepressors modulate AR-induced gene expression in prostate cancer cells and may play a role in development and progression of prostate cancer. Coactivators recruit other proteins such as histone acetylases and other histone modifiers and chromatin remodeling complexes to promote transcription whereas corepressors are proposed to function by binding to unliganded receptor and recruiting histone deacetylases6. The nuclear receptor coactivator gene NCOA2  is frequently amplified in prostate cancer tumors and overexpression of NCOA2 may sensitize AR to be activated by low levels of androgen7. Conversely, expression of nuclear receptor corepressors NCOR1 and NCOR2 is decreased in metastatic prostate cancer, a finding highlighting the potential clinical relevance of androgen receptor corepressor/coactivator balance in prostate cancer8.The AR protein is a steroid receptor composed of three major domains, the N-terminal transactivation domain, the DNA-binding domain, and the C-terminal ligand binding domain10. We hypothesized that Ack1 may affect the proteins interacting with AR and identified SLIRP as a candidate protein whose association with AR is regulated by Ack1. SLIRP (SRA stem-loop interacting RNA binding protein) was initially isolated as a protein binding to the stem-loop structure of the RNA molecule SRA (steroid receptor RNA activator)11. SRA is postulated to act as a long noncoding RNA coactivator of nuclear receptors through its ability to form complexes with coactivators and corepressors13. SLIRP is a small protein (~13 kDa) composed mostly of the RNA recognition motifs and appears to function in nuclear receptor corepressor complexes11. In addition, SLIRP binds to LRPPRC protein in the mitochondria and acts as a global RNA chaperone involved in mitochondrial gene expression16. In this work, we show that SLIRP associating with AR is influenced by Ack1 kinase and androgen, which eject it from the AR complex, modulating AR signaling.Another potential regulatory mechanism for AR activity is crosstalk with tyrosine kinase-dependent pathways. We have demonstrated that phosphorylation of AR at Tyr-267 by Ack1 (TNK2) nonreceptor tyrosine kinase results in nuclear translocation, DNA binding, and transactivation of target genes in the low androgen environmentIn an attempt to identify AR interacting proteins regulated by Ack1 kinase, we employed the differential in-gel electrophoresis (DIGE) approach to compare proteins co-immunoprecipitating with AR in 293 T cells transfected to express AR only versus AR and constitutively active Ack1  induces Src kinase activation and AR phosphorylation at Tyr-534ion Fig. . Treatmeion Fig. . Howeverion Fig. . Loss ofion Fig. .Figure 211. Therefore, we investigated the role of SRA in AR-SLIRP interaction by using the siRNA specific for SRA  in the absence or suboptimal dose of androgen, but less at the full dose  and Ingenuity Pathway analysis (IPA) software to explore biological diseases and pathways that were significantly affected by the loss of SLIRP. DAVID highlighted significantly enriched gene ontology like acetylation which plays a role in epigenetic regulation  in cells transfected with nonsense RNA generated a list of 176 androgen-regulated genes under suboptimal stimulation (Supplementary Table). Hierarchical analysis of this 176 androgen-regulated gene signature applied to the RNA-seq data of all four groups revealed three classes of genes that respond differentially to loss of SLIRP Fig. . The exp18. The finding that association between AR and SLIRP is disrupted by Ack1 is similar to the effect of Src on another AR corepressor LCoR (ligand-dependent corepressor)19. Interaction of LCoR and AR has been shown to be inhibited by Src tyrosine kinase, which activates AR, in part, through phosphorylation of AR at Tyr-53421. Therefore, SLIRP displays properties of a bona fide corepressor of AR. However, similar to many nuclear receptor coregulators, SLIRP affects a broad array of nuclear receptors, including estrogen receptor11. Binding of AR to corepressors NCOR and SMRT involves direct interaction between AR and the corepressor24. In this work, we demonstrated that association between AR and SLIRP requires the SRA RNA molecule. Lanz et al. reported that SRA is found in the ribonucleoprotein complex containing nuclear receptor and that SRA can be co-immunoprecipitated by the AR antibody12. Therefore, we hypothesize that AR-SLIRP interaction may be indirect and the SRA molecule may be a necessary component for bringing AR and SLIRP together into the same complex. Mechanisms by which Ack1 or heregulin leads to dissociation between SLIRP and AR are unclear. It is unlikely to involve direct tyrosine phosphorylation of AR or SLIRP. SLIRP protein does not contain tyrosine residues. AR mutants in which Tyr-267, −363- and −534 have been mutated behave in the same way as wild-type AR. It is postulated that Ack1 kinase-induced or ligand-induced change in the AR conformation leads to assembly of the active AR transcription complex and “exclusion” of SLIRP from this complex. It’s possible that SLIRP may bind (directly or indirectly) to the same site that Ack1 uses to bind to AR. Also, the SLIRP-SRA complex associating with AR may represent a small fraction of the SRA bound to AR. But detailed mechanisms underlying this process remain to be characterized. AR splice variant 7 missing the ligand-bind domain has emerged as a mediator of resistance to androgen signaling inhibitors abiraterone and enzalutamide25. Our data show the lack of interaction between SLIRP and the truncated AR 1–660 mutant . This may provide an additional explanation for the constitutively activated nature of AR-V727.In this report, we show that complex formation between SLIRP and AR can be regulated by androgen or by activated Ack1 kinase and heregulin treatment. In the absence of ligand- or kinase-induced activation, there is a stable complex including SLIRP and AR and presumably the RNA component SRA. Androgen stimulation, Ack1 kinase, and heregulin treatment (presumably by activating the ErbB2 receptor and Ack1 downstream) lead to dissociation of SLIRP from AR. In the absence of androgen, SLIRP is constitutively bound to the enhancer elements of the canonical androgen-regulated genes (e.g. PSA and hK2). After androgen stimulation or treatment with heregulin , AR is recruited to the enhancer while SLIRP dissociates from the enhancer. Other well-characterized AR corepressors NCOR and SMRT display similar behavior as they are recruited to the DNA by bicalutamide but not by androgen28. Liu et al.28 characterized the effect of knocking down 18 clinically relevant AR coregulators on the expression of the 452 AR target gene signature in LNCaP cells. Knockdown of p300 had the broadest impact on gene expression, affecting 57% of AR target genes. Knockdown of NCOA1 and NCOA2 affected only 11% and 10% of genes. Interestingly, knockdown of coregulators simultaneously increased expression of certain genes while decreasing expression of other genes. Our data on SLIRP, in agreement with Liu et al.28, suggest that each AR coregulator affects only a subset of AR target genes. However, since the two studies used different conditions for androgen induction , the specific numbers and proportions of genes may not be comparable. In addition to its role in regulating AR and other nuclear receptors, SLIRP is likely to be involved in several other processes such as mitochondrial gene expression16. We found that SLIRP knockdown affects expression of more than 3000 genes. SLIRP knockout mice are viable, but have minimally reduced fertility29.The role of SLIRP as an AR corepressor is also supported by whole transcriptome analysis showing that expression of the majority (92 out of 176 or 52%) of androgen-regulated genes are increased by loss of SLIRP. The fact that loss of SLIRP increases expression of these genes in the absence of androgen stimulation raises a possibility that loss of SLIRP facilitates nuclear localization and activity of AR. Our data showing that loss of SLIRP had no effect on some AR-responsive genes and led to decreased expression of other androgen-regulated genes are consistent with a recent report on a comprehensive analysis of androgen receptor coregulators in prostate cancerhttp://cbioportal.org)30 for alteration of SLIRP in prostate cancer revealed no point mutation of SLIRP. Analysis of currently available data in cBioPortal showed that 6.9% of primary tumors (84 out of 1121) demonstrated SLIRP gene copy number loss while 11.4% of metastatic tumors (105 out of 918) demonstrated SLIRP gene copy number loss . The role of SLIRP in clinical progression of prostate cancer is uncertain and will require more investigation.The role of SLIRP in prostate cancer progression was investigated by exploratory analysis of clinical specimens. Query of the cBioPortal for Cancer Genomics  was obtained from Millipore . Antibody against SLIRP (#ab51523) was purchased from Abcam . Antibodies against total ERK (#9102) and phospho-ERK (#9101) were obtained from Cell Signaling Technology . Actin antibody (#A3853) and anti-Flag affinity gel (#A2220) were purchased from Sigma-Aldrich .LNCaP cells and 293 T cells were obtained from the American Type Culture Collection . EGF , IL-6 (R&D Systems), Gas6 (R&D Systems) and bombesin , U0126  were purchased. Heregulin was a gift from Genentech . Dasatinib was a gift from Bristol-Myers-Squibb . A mouse monoclonal antibody against AR  was used for immunoblotting and a polyclonal antibody against AR  was used for immunoprecipitation. The antibody against total Ack1 was described previously34. Flag-SLIRP and SRA expressing vector were purchased from Origene Inc. . Y267F, Y363F, Y534F mutants of AR were constructed using Stratagene QuikChange™ Site-Directed Mutagenesis Kit , as previously described35.The plasmids encoding AR, wild-type (wt) Ack1, kinase dead (kd) Ack1, constitutively active (ca) Ack1, ARR2-PB-luciferase reporter were previously described10. Briefly, an antibody against AR  or SLIRP  was applied to immunoprecipitate DNA which is associated with AR and SLIRP. DNA was subjected to quantitative PCR using the primers and the probe targeting the distal ARE III enhancer sequence of the PSA gene or the primers and the probe targeting the distal enhancer of the hK2 gene as described previously10.Cells were lysed in lysis buffer containing 50 mmol/L Tris-HCl, 0.1% NP40, 150 mmol/L NaCl, 10% glycerol, 2 mmol/L EDTA, plus proteinase inhibitor  and phosphatase inhibitor . Immunoprecipitation was done by incubating the mixture of 500 μg protein lysis with 2 μg IgG and 50 μL protein A agarose beads  overnight at 4 °C. Immunoprecipitated fraction was resolved on 4–12% Bis-tris gel . For immunoblottting, antibodies were prepared at 1:1000 dilution unless specifically indicated. ChIP analysis was performed following the protocol described beforeBoth LNCaP cells and 293 T cells were transfected with expression vectors encoding AR, Ack1, or Flag-SLIRP using Effectene  according to the manufacturer’s instructions. To knock down SLIRP and SRA, validated Stealth RNAi siRNA against SLIRP and SRA  was used according to the manufacturer’s instructions. 24 h after transfection, cells were treated with ligands in serum-free media as indicated.36. The spot of interested was picked and analyzed by mass spectrometry at the UNC Proteomics Core Facility. The resulting peptides were mixed with matrix (α-Cyano-4-Hydroxycinnamic Acid) and analyzed using a MALDI-TOF/TOF mass spectrometer (Applied Biosystems 4800 Plus). MS spectra were obtained in reflector positive ion mode and peaks with signal-to-noise ratio above 20 were selected for MS/MS analysis (maximum of 45 MS/MS spectra per spot). All spectra were searched using GPS Explorer Software Version 3.6 (Applied Biosystems) linked to the Mascot  search engine.293 T cells in 10 cm culture dish were transfected with the expression vectors encoding AR (1 μg), or AR (1 μg) plus constitutively active Ack1 (1 μg) using Effectene. After 24 h, protein extracts were harvested and immunoprecipiated with AR antibody as described above. The precipitant was analyzed using one-dimensional DIGE method in UNC Systems-Proteomics Core Facility, following protocols described previously4 cells per well/12well plate) were transfected with the ARR2-PB-luciferase reporter (0.1 μg) along with the AR expression vector (0.1 μg), and vector (3 μg) or SLIRP (3 μg) using Effectene, as described previously9. After overnight incubation, cells were pretreated with 1 nM DHT as indicated for 24 h. Luciferase activity was determined as described previously. For measurement of PSA and hK2 mRNA levels, LNCaP cells were transfected with 40 nM siRNA using Effectene for 24 h. Next day, the cells were treated with 1 nM DHT in serum-free medium for 24 h. Total RNA was isolated and the mRNA levels of PSA and hK2 were determined by quantitative reverse-transcription PCR, as described previously10. To detect the level of SRA RNA, total RNA was isolated using TRI Reagent  per manufacturer’s instruction. First-strand cDNA was synthesized using oligo-dT primers. Quantitative PCR analysis was performed in triplicate using Cybergreen master mix  in the ABI PRISM 7900HT system . The primer sequence were obtained from Agoulnik et al.: forward, 5′TCTACTGGTGCAAGAGCTTTCAAG3′; reverse, 5′ATGAGGGAGCGGTGGATGT3′37.LNCaP cells . Genelists were also generated for AR signature using SAM by comparing NS-vehicle versus NS-1nM DHT. Heatmap was generated by using Cluster (Stanford University) and Java Treeview (Sourceforge). Sequencing data are available in the Gene Expression Omnibus database under the accession number GSE77829.LNCaP cells were transfected with custom nonsense control siRNA (sense-GUUCAGGUCGAUAUGUGCA) or a pool of SLIRP siRNA  at 40 nM for 24 hrs. Cells were washed once with phosphate-buffered saline and replaced with serum-free medium containing EtOH or 1 nM DHT for another 24 hrs. RNA was collected using RNeasy kit . RNA concentration was checked by Nanodrop and quality assayed by Bioanalyzer. Two biological replicates were collected from 2 different experiments for a total of 4 replicates. RNA collected was sequenced using Illumia HiSeq. 2000 single read 50 base pair by the UNC High Throughput Sequencing Facility (reference genome hg19) and aligned and normalized by the Bioinformatics core. Resulting dataset was analyzed by SAM and the up/down-regulated gene list generated with an FDR ≤ 1 were used in DAVID functional softwaret test, with a p value of <0.05 regarded as significant.Transfection and luciferase data are shown as mean ± SEM. Statistical analysis was performed using Student’s Supplementary TableSupplementary Information